Polypeptide having alpha-isomaltosylglucosaccharide synthase activity

ABSTRACT

The object of the present invention is to provide a polypeptide which can be used to produce a saccharide having the structure of cyclo{→6)-α-D-glucopyranosyl-(1→3)-α-D-glucopyranosyl-(1→6)-α-D-glucopyranosyl-(1→3)-α-D-glucopyranosyl-(1→}, a DNA encoding the polypeptide, and uses thereof. The present invention solves the above object by establishing a polypeptide which has an enzymatic activity of forming a saccharide with a glucose polymerization degree of 3 or higher and bearing both the α-1,6 glucosidic linkage as a linkage at the non-reducing end and the α-1,4 glucosidic linkage other than the linkage at the non-reducing end from a saccharide with a glucose polymerization degree of 2 or higher and bearing the α-1,4 glucosidic linkage as a linkage at the non-reducing end by α-glucosyl transferring reaction without substantially increasing the reducing power, a DNA encoding the polypeptide, a replicable recombinant DNA comprising the DNA encoding the polypeptide and an autonomously replicable vector, a transformant constructed by introducing the recombinant DNA into an appropriate host, and uses thereof.

TECHNICAL FIELD

[0001] The present invention relates to a polypeptide which has anactivity of forming α-isomaltosylglucosaccharides (which may be simplydesignated as “a polypeptide”), a process for preparing the polypeptideby gene recombinant technology, and uses thereof.

BACKGROUND ART

[0002] There have been known several carbohydrates which are composed ofglucose molecules as constituents and produced from starches, amyloses,or partial starch hydrolyzates as amylaceous materials, for example,amylodextrins, maltodextrins, maltooligosaccharides, andisomaltooligosaccharides. These carbohydrates are also known to haveusually both non-reducing and reducing groups at their molecular endsand the reducing group is responsible for reducibility. In general,reducing power of a partial starch hydrolyzate is represented withdextrose equivalent (DE), a scale for the reducing power, on a dry solidbasis. Such a partial starch hydrolyzate with a high DE value has a lowmolecular weight, viscosity, strong sweetening power and reactivity:They easily react with amino group-containing substances such as aminoacids and proteins through the amino carbonyl reaction which may lead tobrowning, undesirable smell, and deterioration. To overcome thesedisadvantages, heretofore long desired are methods which may lower oreven eliminate the reducing power of partial starch hydrolyzates withoutconverting glucose molecules as constituent saccharides. For example, itwas reported in Journal of the American Chemical Society, Vol. 71,353-358 (1949) that starches can be converted to α-, β- andγ-cyclodextrins which are composed of 6-8 glucose molecules linkedcovalently via the α-1,4 glucosidic linkage by allowing to contact with“macerans amylase”. Nowadays, cyclodextrins are produced on anindustrial scale and their inherent properties such as non-reducibility,tasteless, and clathrating abilities render them very useful in avariety of fields. While, for example, Japanese Patent Kokai Nos.143,876/95 and 213,283/95, filed by the same applicant of the presentinvention discloses a method of producing trehalose, a disaccharidecomposed of two glucose molecules linked together via the α,α-linkage,where a non-reducing saccharide-forming enzyme and a trehalose-releasingenzyme are allowed to contact with partial starch hydrolyzates such asmaltooligosaccharides. In these days, trehalose has been industriallyproduced from starches and applied to a variety of fields where itsnon-reducibility, mild- and high quality-sweetness are advantageouslyutilized. As described above, trehalose (DP of two) and α-, β- andγ-cyclodextrins (DP of 6-8) have been produced on an industrial scaleand extensively used because of their advantageous properties; however,there is a limitation in the types of non- or low-reducing saccharideswhich are available in the art. Therefore, saccharides other than thesesaccharides are in great demand.

[0003] Recently, reported was a novel cyclic tetrasaccharide composed ofglucose units: European Journal of Biochemistry, Vol.226, 641-648 (1994)reported that a cyclic tetrasaccharide with the structure ofcyclo{→6)-α-D-glucopyranosyl-(1→3)-α-D-glucopyranosyl-(1→6)-α-D-glucopyranosyl-(1→3)-α-D-glucopyranosyl-(1→} (which may be simply designated as“cyclotetrasaccharide”, hereinafter) is formed by allowing alternanase,a type of hydrolyzing enzyme, to contact with alternan, a polysaccharidewhere glucose molecules are linked via the alternating α-1,3 and α-1,6bonds, followed by crystallization in the presence of methanol.

[0004] Cyclotetrasaccharide, a saccharide with a cyclic structure and noreducing power, is expected to be very useful because of its noamino-carbonyl reactivity, stabilizing effect for volatile organiccompounds by its clathrating ability, and no apprehension of browningand deterioration.

[0005] However, alternan and alternanase, which are indispensablematerials to produce cyclotetrasaccharide, are not easily obtainable,and microorganisms as alternanase source are not easily available.

[0006] Under these circumstances, the present inventors disclosed in WO01/90338 Al a successful process to produce cyclotetrasaccharide where asaccharide with a glucose polymerization degree of 3 or higher andbearing both the α-1,6 glucosidic linkage as a linkage at thenon-reducing end and the α-1,4 glucosidic linkage other than the linkageat the non-reducing end (may be called “α-isomaltosylglucosaccharide”throughout the specification) as a material is allowed to contact withan α-isomaltosyl-transferring enzyme which specifically hydrolyzes thelinkage between the α-isomaltosyl moiety and the resting glucosaccharidemoiety, and then the enzyme transfers the released α-isomaltosyl moietyto another α-isomaltosylglucosaccharide to form cyclotetrasaccharide.The α-isomaltosyl-transferring enzyme forms cyclotetrasaccharide fromα-isomaltosylglucosaccharide by α-isomaltosyl-transferring reaction.Particularly, α-isomaltosyl-transferring enzyme has the followingphysicochemical properties:

[0007] (1) Action

[0008] Forming cyclotetrasaccharide with the structure ofcyclo{→6)-α-D-glucopyranosyl-(1→3)-α-D-glucopyranosyl-(1→6)-α-D-glucopyranosyl-(13)-α-D-glucopyranosyl-(1→} from a saccharide with a glucosepolymerization degree of 3 or higher and bearing both the α-1,6glucosidic linkage as a linkage at the non-reducing end and the α-1,4glucosidic linkage other than the linkage at the non-reducing end bycatalyzing α-isomaltosyl-transferring reaction;

[0009] (2) Molecular weight

[0010] About 82,000 to 136,000 daltons when determined on sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE);

[0011] (3) Isoelectric point (pI)

[0012] About 3.7 to 8.3 when determined on isoelectrophoresis usingampholine;

[0013] (4) Optimum temperature

[0014] About 45 to 50° C. when incubated at pH 6.0 for 30 minutes;

[0015] (5) Optimum pH

[0016] About 5.5 to 6.5 when incubated at 35° C. for 30 minutes;

[0017] (6) Thermal stability

[0018] About 45° C. or lower when incubated at pH 6.0 for 60 minutes;and

[0019] (7) pH Stability

[0020] About 3.6 to 10.0 when incubated at 4° C. for 24 hours.

[0021] As regards to saccharides which are used as starting materialsfor cyclotetrasaccharide, it is desirable to prepare them from starcheswhich are abundant and low-cost sources, however, sinceα-isomaltosyl-transferring enzyme dose not directly act on starches, thefollowing procedures are actually employed: Starches are firstlyconverted into an α-isomaltosylglucosaccharide having the abovespecified structure, for example, relatively low-molecular weightisomaltooligosaccharide such as panose and isomaltosylmaltose, and thensubjected to the action of α-isomaltosyl-transferring enzyme to formcyclotetrasaccharide. As regards to the yield of cyclotetrasaccharidefrom the materials, in the case of using panose as a material, the yieldof cyclotetrasaccharide is about 44% based on the weight of the drysolid (d.s.b.). Similarly, in the case of using isomaltosylmaltose as amaterial, the yield of cyclotetrasaccharide is about 31%, d.s.b. Whilein the case of using starches as a material, it is necessary to contactstarches previously with α-amylase, starch-debranching enzyme, β-amylaseand α-glucosidase to form relatively low-molecular weightisomaltooligosaccharides including panose, and the yield ofcyclotetrasaccharide is relatively low, about 15%, d.s.b. Although theproduction of cyclotetrasaccharide from starch is feasible even in sucha low yield, the production cost may be increased. Therefore, it isdesired to establish a novel method for producing cyclotetrasaccharidein a relatively high yield using starches as a material.

[0022] Under these circumstances, the present inventors extensivelyscreened microorganisms capable of producing anα-isomaltosylglucosaccharide-forming enzyme which may significantlyimprove the yield of cyclotetrasaccharide when allowed to act onstarches as a material. As a result, the present inventors found thatα-isomaltosyl-transferring enzyme-producing microorganisms, strains C9,C11, N75 and A19 of the genera Bacillus and Arthrobacter, which aredisclosed in WO 01/90338 A1, also produce anotherα-isomaltosylglucosaccharide-forming enzyme. They also found that theyield of cyclotetrasaccharide can be remarkably improved by allowingboth α-isomaltosylglucosaccharide-forming enzyme andα-isomaltosyl-transferring enzyme to act on a glucosaccharide with ahigh-molecular weight such as partial starch hydrolyzates. The presentinventors characterized the α-isomaltosylglucosaccharide-forming enzyme,and established a process for producing the enzyme. Further, theyestablished methods for α-glucosyl-transferring reaction using theenzyme, a process for producing α-isomaltosylglucosaccharide, and aprocess for producing cyclotetrasaccharide and a saccharide compositioncontaining the cyclotetrasaccharide by the combination use of the enzymeand α-isomaltosyl-transferring enzyme. Also, the present inventorsestablished food products, cosmetics and pharmaceuticals, comprisingcyclotetrasaccharide which are obtainable by the processes mentionedabove or saccharide compositions containing cyclotetrasaccharide.However, since the producibility of α-isomaltosylglucosaccharide-formingenzyme in the microorganisms were found to be not enough, there has beenstill left a problem that large-scale cultivation of such microorganismsas enzyme sources are required for industrial scale production ofα-isomaltosylglucosaccharide and cyclotetrasaccharide.

[0023] It is known that the entity of the enzyme is a polypeptide andthe enzymatic activity is under the regulation of its amino acidsequence, which is encoded by a DNA. Therefore, if one successfullyisolates a gene which encodes the polypeptide and determines thenucleotide sequence, it will be relatively easy to obtain the desiredamount of the polypeptide by the steps of constructing a recombinant DNAwhich contains the DNA encoding the polypeptide, introducing therecombinant DNA into host-cells such as microorganisms, animals orplants, and culturing the obtained transformants in appropriate nutrientmedia. Under these, required are the isolation of a gene encoding thepolypeptide as the entity of the enzyme described above, and thesequencing of the nucleotide sequence.

DISCLOSURE OF INVENTION

[0024] The first object of the present invention is to establish apolypeptide which has an α-isomaltosylglucosaccharide-forming enzymeactivity of catalyzing the formation of a saccharide with a glucosepolymerization degree of 3 or higher and bearing both the α-1,6glucosidic linkage as a linkage at the non-reducing end and the α-1,4glucosidic linkage other than the linkage at the non-reducing end, froma saccharide with a glucose polymerization degree of 2 or higher andbearing the α-1,4 glucosidic linkage as a linkage at the non-reducingend by α-glucosyl transferring reaction without substantially increasingthe reducing power.

[0025] The second object of the present invention is to provide a DNAencoding the above polypeptide.

[0026] The third object of the present invention is to provide areplicable recombinant DNA comprising the above DNA.

[0027] The fourth object of the present invention is to provide atransformant transformed by the recombinant DNA.

[0028] The fifth object of the present invention is to provide a processfor producing the polypeptide havingα-isomaltosylglucosaccharide-forming enzyme activity by using thetransformant.

[0029] The sixth object of the present invention is to provide uses ofthe polypeptide described above.

[0030] The present invention solves the first object described above byproviding a polypeptide having α-isomaltosylglucosaccharide-formingactivity and the following physicochemical properties:

[0031] (1) Forming a saccharide with a glucose polymerization degree of3 or higher and bearing both the α-1,6 glucosidic linkage as a linkageat the non-reducing end and the α-1,4 glucosidic linkage other than thelinkage at the non-reducing end from a saccharide with a glucosepolymerization degree of 2 or higher and bearing the α-1,4 glucosidiclinkage as a linkage at the non-reducing end by α-glucosyl transferringreaction without substantially increasing the reducing power;

[0032] (2) Molecular weight

[0033] About 74,000 to 160,000 daltons when determined on sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE);

[0034] (3) Optimum temperature

[0035] About 40° C. to 50° C. when incubated at pH 6.0 for 60 minutes;

[0036] About 45° C. to 55° C. when incubated at pH 6.0 for 60 minutes inthe presence of 1 mM Ca²⁺;

[0037] About 60° C. when incubated at pH 8.4 for 60 minutes; or

[0038] About 65° C. when incubated at pH 8.4 for 60 minutes in thepresence of 1 mM Ca²⁺;

[0039] (4) Optimum pH

[0040] About 6.0 to 8.4 when incubated at 35° C. for 60 minutes;

[0041] (5) Thermal stability

[0042] About 45° C. or lower when incubated at pH 6.0 for 60 minutes;

[0043] About 60° C. or lower when incubated at pH 6.0 for 60 minutes inthe presence of 1 mM Ca²⁺;

[0044] About 55° C. or lower when incubated at pH 8.0 for 60 minutes; or

[0045] About 60° C. or lower when incubated at pH 6.0 for 60 minutes inthe presence of 1 mM Ca²⁺; and

[0046] (6) pH Stability

[0047] About 5.0 to 10.0 when incubated at 4° C. for 24 hours.

[0048] The present invention solves the second object described above byproviding a DNA encoding the polypeptide.

[0049] The present invention solves the third object described above byproviding a replicable recombinant DNA which comprise a DNA encoding thepolypeptide and an autonomously replicable vector.

[0050] The present invention solves the fourth object described above byproviding a transformant constructed by introducing the recombinant DNAinto an appropriate host.

[0051] The present invention solves the fifth object described above byproviding a process for preparing the polypeptide, which comprises thesteps of culturing a transformant constructed by introducing areplicable recombinant DNA, which contains a DNA encoding thepolypeptide and an autonomously replicable vector, into an appropriatehost; and collecting the polypeptide from the resultant culture.

[0052] The present invention solves the sixth object described above byestablishing various uses of the polypeptide.

BRIEF DESCRIPTION OF DRAWINGS

[0053]FIG. 1 shows a ¹H-NMR spectrum of an enzymatic reaction product Xwhich was obtained from maltotetraose using a polypeptide havingα-isomaltosylglucosaccharide-forming enzyme activity.

[0054]FIG. 2 shows a ¹H-NMR spectrum of an enzymatic reaction product Ywhich was obtained from maltopentaose using a polypeptide havingα-isomaltosylglucosaccharide-forming enzyme activity.

[0055]FIG. 3 shows a ¹³C-NMR spectrum of an enzymatic reaction product Xwhich was obtained from maltotetraose using a polypeptide havingα-isomaltosylglucosaccharide-forming enzyme activity.

[0056]FIG. 4 shows a ¹³C-NMR spectrum of an enzymatic reaction product Ywhich was obtained from maltopentaose using a polypeptide havingα-isomaltosylglucosaccharide-forming enzyme activity.

[0057]FIG. 5 shows the effect of temperature onα-isomaltosylglucosaccharide-forming enzyme activity from amicroorganism of Bacillus globisporus C11.

[0058]FIG. 6 shows the effect of temperature onα-isomaltosylglucosaccharide-forming enzyme activity from amicroorganism of Bacillus globisporus N75.

[0059]FIG. 7 shows the effect of temperature onα-isomaltosylglucosaccharide-forming enzyme activity from amicroorganism of Arthrobacter globiformis A19.

[0060]FIG. 8 shows the effect of pH onα-isomaltosylglucosaccharide-forming enzyme activity from amicroorganism of Bacillus globisporus C11.

[0061]FIG. 9 shows the effect of pH onα-isomaltosylglucosaccharide-forming enzyme activity from amicroorganism of Bacillus globisporus N75.

[0062]FIG. 10 shows the effect of pH onα-isomaltosylglucosaccharide-forming enzyme activity from amicroorganism of Arthrobacter globiformis A19.

[0063]FIG. 11 shows the thermal stability ofα-isomaltosylglucosaccharide-forming enzyme from a microorganism ofBacillus globisporus C11.

[0064]FIG. 12 shows the thermal stability ofα-isomaltosylglucosaccharide-forming enzyme from a microorganism ofBacillus globisporus N75.

[0065]FIG. 13 shows the thermal stability ofα-isomaltosylglucosaccharide-forming enzyme from a microorganism ofArthrobacter globiformis A19.

[0066]FIG. 14 shows the pH stability ofα-isomaltosylglucosaccharide-forming enzyme from a microorganism ofBacillus globisporus C11.

[0067]FIG. 15 shows the pH stability ofα-isomaltosylglucosaccharide-forming enzyme from a microorganism ofBacillus globisporus N75.

[0068]FIG. 16 shows the. pH stability ofα-isomaltosylglucosaccharide-forming enzyme from a microorganism ofArthrobacter globiformis A19.

[0069]FIG. 17 shows a restriction enzyme map of a recombinant DNA,pBGC2, of the present invention. In the figure, a section indicated witha black bold line is a DNA encoding a polypeptide havingα-isomaltosyl-transferring enzyme activity from a microorganism ofBacillus globisporus C11.

[0070]FIG. 18 shows a restriction enzyme map of a recombinant DNA,pBGN2, of the present invention. In the figure, a section indicated witha black bold line is a DNA encoding a polypeptide havingα-isomaltosyl-transferring enzyme activity from a microorganism ofBacillus globisporus N75.

[0071]FIG. 19 shows a restriction enzyme map of a recombinant DNA,pAGA1, of the present invention. In the figure, a section indicated witha black bold line is a DNA encoding a polypeptide havingα-isomaltosyl-transferring enzyme activity from a microorganism ofArthrobacter globiformis A19.

BEST MODE FOR CARRYING OUT THE INVENTION

[0072] The wording “polypeptide” as referred to as in the presentinvention means polypeptides in general which have an activity offorming a saccharide with a glucose polymerization degree of 3 or higherand bearing both the α-1,6 glucosidic linkage as a linkage at thenon-reducing end and the α-1,4 glucosidic linkage other than the linkageat the non-reducing end from a saccharide with a glucose polymerizationdegree of 2 or higher and bearing α-1,4 glucosidic linkage as a linkageat the non-reducing end by α-glucosyl-transferring reaction withoutsubstantially increasing the reducing power.

[0073] The polypeptide of the present invention usually comprises adetermined amino acid sequence, for example, an amino acid sequence ofSEQ ID NO:1 or mutants of SEQ ID NO:1 having deletion, replacement withdifferent amino acid(s), or addition of one or more of amino acids,i.e., at least one or two, for example, 1-50, 1-30, or 1-10 amino acidsof SEQ ID NO:1. Even-with the same DNA, the post-translationalmodification of a polypeptide by extra-/intra-cellular enzymes of hostis affected by various conditions such as the kind of host, nutrients orcomposition of culture media, temperatures or pHs for the cultivation ofa transformant having the above DNA. In such conditions, it is possibleto arise some mutants having deletion or replacement with differentamino acid of one or more, i.e., at least one or two, according to thesituation, 1-30, 1-20, or 1-10 amino acids of the N-terminal region ofSEQ ID NO: 1, further, or having addition of one or more, i.e., at leastone or two, for example, 1-30, 1-20, or 1-10 amino acids to thoseN-termini. The -polypeptide of the present invention includes thesemutants as far as their physicochemical properties have been revealed.

[0074] The polypeptide of the present invention can be obtained by thesteps of introducing the DNA of the present invention into appropriatehosts, and collecting the polypeptide from the culture of the resultanttransformants. The transformants usable in the present invention arethose which contain a DNA comprising, for example, a nucleotidesequence, from the 5′-terminus, of SEQ ID NO:4, SEQ ID NO:5, or SEQ IDNO:6; those having deletion, replacement or addition of one or morenucleotides in the above nucleotide sequence, those having anti-sensenucleotide sequences, or those having replacement of one or morenucleotides based on the degeneracy of genetic code without altering theamino acid sequence encoded by the above nucleotide sequence. Thenucleotide sequence having replacement of one or more, i.e., at leastone or two, optionally, 1-150, 1-90, 1-60, or 1-30 nucleotides based ongene-degeneracy without altering the amino acid sequence encoded by theabove nucleotide sequence can be used as the nucleotide sequence.

[0075] The DNAs of the present invention include those of natural originor those which can be synthesized in an artificial manner can be used asfar as the DNAs encode the polypeptide of the present invention can beobtained. Microorganisms of the genus Bacillus, for example, Bacillusglobisporus C9, deposited on Apr. 25, 2000, in the InternationalDepositary Authority National Institute of Bioscience andHuman-Technology, Agency of Industrial Science and Technology, 1-3,Higashi 1 chome Tsukuba-shi, Ibaraki-ken, Japan (International PatentOrganism Depositary National Institute of Advanced Industrial Scienceand Technology Tsukuba Central 6, 1-1, Higashi 1-Chome Tsukuba-shi,Ibaraki-ken, 305-8566, Japan) under the accession number of FERMBP-7143; Bacillus globisporus C11, deposited on Apr. 25, 2000, in theInternational Patent Organism Depositary National Institute of AdvancedIndustrial Science and Technology Tsukuba Central 6, 1-1, Higashi1-Chome Tsukuba-shi, Ibaraki-ken, 305-8566, Japan under the accessionnumber of FERM BP-7144; and Bacillus globisporus N75, deposited on May16, 2001, in the International Patent Organism Depositary NationalInstitute of Advanced Industrial Science and Technology Tsukuba Central6, 1-1, Higashi 1-Chome Tsukuba-shi, Ibaraki-ken, 305-8566, Japan underthe accession number of FERMBP-7591; and other microorganisms belongingthe genus Arthrobacter including Arthrobacter globiformis A19, depositedon May 16, 2001, in the International Patent Organism DepositaryNational Institute of Advanced Industrial Science and Technology TsukubaCentral 6, 1-1, Higashi 1-Chome Tsukuba-shi, Ibaraki-ken, 305-8566,Japan under the accession number of FERM BP-7590, are usable as thenatural sources. A gene containing the DNA of the present invention canbe obtained from the cells of the above microorganisms. Specifically, agene containing the DNA can be released extracellularly by the steps ofinoculating the microorganisms into a nutrient medium, culturing themabout for one to three days under aerobic conditions, collecting theproliferated cells from the culture, and treating the cells withcell-lysis enzymes such as lysozyme and β-glucanase or withultrasonication. In addition to the above methods, protein-hydrolyzingenzymes such as proteinases and freeze-thawing method in the presence ofdetergents such as sodium dodecyl sulfate can be used. The objectiveDNAs can be obtained from the disrupted cells using conventional methodsin the art, for example, phenol-extraction, alcohol-precipitation,centrifugation, and ribonuclease-treatment. To synthesize the DNAs ofthe present invention artificially, chemical synthesis of the DNAs usingthe nucleotide sequence of SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:6 canbe used. Also, PCR-method can be advantageously used to obtain the DNAsusing a gene containing them as a template and appropriate chemicallysynthetic DNA as a primer.

[0076] The use of such DNAs enables the industrial production of thepolypeptide of the present invention in a large amount and at arelatively low-cost: Such a production usually comprises the steps ofinserting a specific DNA into an appropriate autonomously replicablevector to construct a recombinant DNA, introducing the resultantrecombinant DNA into an appropriate host, culturing the resultanttransformant in an appropriate nutrient medium for proliferation,collecting the cells from the culture, collecting the recombinant DNAfrom the cells, introducing the recombinant DNA into an appropriate hostwhich can be easily proliferated for transformation, and culturing theresultant transformant in an appropriate nutrient medium. If one canobtain a DNA encoding the polypeptide of the present invention, therecombinant DNA described above can be relatively easily prepared byconventional recombinant DNA techniques. For example, plasmid vectorssuch as pBR322, pUC18, Bluescript II SK(+), pUB110, pTZ4, pC194, pHV14,TRp7, YEp7 and pBS7; or phage vectors such as λgt·λC, λgt·λB, ρ11, φ1and φ105 can be used as the vectors. To express the DNA of the presentinvention in E. coli, pBR322, pUC18, Bluescript II SK(+), λgt·λC, andλgt·λB are preferable. To express the DNA of the present invention inBacillus subtilis, pUB110, pTZ4, pC194, ρ11, φ1 and φ105 are preferable.Plasmids, pHV14, TRp7, YEp7 and pBS7 are useful in the case ofreplicating the recombinant DNA of the present invention in two or morehosts.

[0077] In order to insert the DNAs of the present invention into thesevectors, conventional methods used in the art can be used. Specifically,a specific DNA is inserted into a vector by the steps of cleaving a genecontaining the DNA and an autonomously replicable vector by restrictionenzymes and/or ultrasonication and ligating the resulting DNA fragmentand the resulting vector fragment. The ligation of the DNA fragment andthe vector fragment can be easy by using restriction enzymes whichspecifically act on nucleotides, particularly, type II-restrictionenzymes such as Sau 3AI, Eco RI, Hind III, Bam HI, Sal I, Xba I, Sac Iand Pst I for cleaving gene and vector. After the annealing of the both,if necessary, the desired recombinant DNA can be obtained by ligatingthem in vivo or in vitro using a DNA ligase. The recombinant DNA thusobtained is unlimitedly replicable by the steps of introducing intoappropriate hosts such as Escherihia coli, Bacillus subtilis,Actinomyces, and yeasts to construct transformants, and culturing theresultant transformants. On the cloning described above, the desiredclones can be obtained from the transformants by applying thecolony-hybridization method or screening by the steps of culturing in anutrient medium containing saccharides with a glucose polymerizationdegree of 2 or higher and bearing the α-1,4 glucosidic linkage residueas a linkage at the non-reducing end and selecting a trabsformant whichproduces cyclotetrasaccharide from the saccharides.

[0078] The transformant, obtained by the cloning, produces thepolypeptide extra- or intra-cellularly when cultured in a nutrientmedium. Usually, conventional liquid media supplemented with carbonsources, nitrogen sources and minerals, and optionally trace-nutrientssuch as amino acids and vitamins are usually used as the nutrient media.Examples of the carbon sources usable in the present invention aresaccharides including starch, starch hydrolyzate, glucose, fructose,sucrose, and trehalose. Examples of the nitrogen sources usable in thepresent invention are nitrogen-containing inorganic- ororganic-substances including ammonia, ammonium salts, urea, nitrate,peptone, yeast extract, defatted soybean, corn-steep liquor and meatextract. Cultures containing the polypeptide can be obtained by thesteps of inoculating the transformants into the nutrient media,culturing for about one to six days under aerobic conditions such asaeration and agitation conditions while keeping the temperature and pH,usually, at 20-40° C. and pH 2-10. Although the culture can be usedintact as a crude polypeptide preparation comprising the polypeptide ofthe present invention, the polypeptide is usually separated from cellsor cell debris and purified before use; it can be purified from theculture by removing cells or cell debris from the culture and applyingconventional procedures used in the art for purifying polypeptides, forexample, appropriately combining of one or more procedures such asconcentration, salting out, dialysis, precipitation, gel filtrationchromatography, ion-exchange chromatography, hydrophobic chromatography,affinity chromatography, gel electrophoresis and isoelectrophoresis.

[0079] The polypeptide of the present invention has an activity offorming a saccharides with a glucose polymerization degree of 3 orhigher and bearing both the α-1,6-glucosidic linkage at the non-reducingend and the α-1,4 glucosidic linkage other than the linkage at thenon-reducing end from a saccharide with a glucose polymerization degreeof 2 or higher and bearing the α-1,4 glucosidic linkage as a linkage atthe non-reducing end by α-glucosyl transferring reaction withoutsubstantially increasing the reducing power, and has the followingphysicochemical properties:

[0080] (1) Action

[0081] Forming a saccharide with a glucose polymerization degree of 3 orhigher and bearing both the α-1,6 glucosidic linkage as a linkage at thenon-reducing end and the α-1,4 glucosidic linkage other than the linkageat the non-reducing end from a saccharide with a glucose polymerizationdegree of 2 or higher and bearing the α-1,4 glucosidic linkage as alinkage at the non-reducing end by α-glucosyl transferring reactionwithout substantially increasing the reducing power;

[0082] (2) Molecular weight

[0083] About 74,000 to 160,000 daltons when determined on sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE);

[0084] (3) Optimum temperature

[0085] About 40° C. to 50° C. when incubated at pH 6.0 for 60 minutes;

[0086] About 45° C. to 55° C. when incubated at pH 6.0 for 60 minutes inthe presence of 1 mM Ca²⁺;

[0087] About 60° C. when incubated at pH 8.4 for 60 minutes; or

[0088] About 65° C. when incubated at pH 8.4 for 60 minutes in thepresence of 1 mM Ca²⁺;

[0089] (4) Optimum pH

[0090] About 6.0 to 8.4 when incubated at 35° C. for 60 minutes;

[0091] (5) Thermal stability

[0092] About 45° C. or lower when incubated at pH 6.0 for 60 minutes;

[0093] About 60° C. or lower when incubated at pH 6.0 for 60 minutes inthe presence of 1 mM Ca²⁺;

[0094] About 55° C. or lower when incubated at pH 8.0 for 60 minutes; or

[0095] About 60° C. or lower when incubated at pH 6.0 for 60 minutes inthe presence of 1 mM Ca²⁺; and

[0096] (6) pH Stability

[0097] About 5.0 to 10.0 when incubated at 4° C. for 24 hours.

[0098] Polysaccharides comprising the α-1,4 glucosidic linkages such asstarch, amylopectin, amylose, and glycogen, and their partialhydrolyzates such as amylodextrins, maltodextrins andmaltooligosaccharides, which can be obtained by partially hydrolyzingthem with amylases or acids, can be used as a substrate for thepolypeptide of the present invention. Saccharides obtained by treatingthese glucosaccharides having the α-1,4 glucosidic linkage withbranching enzymes (EC 2.4.1.18) can be optionally used as the substrate.The partial hydrolyzates obtained by hydrolyzing with amylases such asα-amylase (EC 3.2.1.1), β-amylase (EC 3.2.1.2), maltotriose-formingamylase (EC 3.2.1.116), maltotetraose-forming amylase (EC 3.2.1.60),maltopentaose-forming amylase, and maltohexaose-forming amylase (EC3.2.1.98), which are described in Handbook of Amylases and RelatedEnzymes, Pergamon Press, Tokyo, Japan (1988), can be used as thesubstrate. Further, in the case of preparing the partial hydrolyzate,starch-debranching enzymes such as pullulanase (EC 3.2.1.41) andisoamylase (EC 3.2.1.68) can be arbitrarily used. Starches as thesubstrates include terrestrial starches from grains such as corn, wheat,and rice; and subterranean starches such as potato, sweet-potato, andtapioca. Preferably, these starches are gelatinized and/or liquefiedinto a liquid form in use. The lower the degree of partial hydrolysis,the higher the yield of cyclotetrasacchaside becomes, and therefore theDE is set to a level of about 20 or lower, preferably, about 12 orlower, more preferably, about five or lower. The concentration of thesubstrates is not specifically restricted, and the enzymatic reaction inthe present invention proceeds even when used at a low concentration aslow as 0.1% (w/w) (throughout the specification, “% (w/w)” isabbreviated as “%' hereinafter, unless specified otherwise). However,one percent or higher concentrations of the substrates are preferablyused for industrial production. The substrate solutions may be those ina suspension form which contain incompletely-dissolved insolublesubstrates. The substrate concentration is preferably 40% or lower, andmore preferably 20% or lower. The reaction temperatures used in thepresent invention are those which proceed the enzymatic reaction, i.e.,those up to 65° C., preferably, 30 to 50° C. The pHs for the enzymaticreaction are usually set to 4.5-8, preferably, about 5.5 to about 7. Thetime for the enzymatic reaction can be appropriately selected dependingon the enzymatic reaction efficiency.

[0099] By contacting α-isomaltosyl-transferring enzyme withα-isomaltosylglucosaccharide formed by acting the polypeptide of thepresent invention on its substrate, cyclotetrasaccharide, which isuseful in the art, can be easily produced in a large amount. Theα-isomaltosyl-transferring enzyme can be allowed to act on its substrateafter the action and then the inactivation of the polypeptide of thepresent invention. However, the combinational use of the polypeptide ofthe present invention and α-isomaltosyl-transferring enzyme ispreferable. Specifically, by using the polypeptide of the presentinvention together with α-isomaltosyl-transferring enzyme,cyclotetrasaccharide can be obtained in a yield of about 30%, d.s.b., orhigher from starches or partial hydrolyzates thereof, and about 80%,d.s.b., or higher from glycogen. The formation mechanism ofcyclotetrasaccharide by the above combinational use can be estimated asfollows based on the reaction properties of the two enzymes:

[0100] (1) The polypeptide of the present invention acts on the α-1,4glucosyl residue at the non-reducing end of a saccharide, which has aglucose polymerization degree of 2 or higher and has the α-1,4glucosidic linkage as a linkage at the non-reducing end, such as starch,glycogen, and the partial hydrolyzates thereof, to release a glucoseresidue; and then intermolecularly transfers the released glucoseresidue to the hydroxyl group at C-6 position of the glucose residue atthe non-reducing end of other saccharide and forms a saccharide havingan α-isomaltosyl residue at the non-reducing end;

[0101] (2) The α-isomaltosyl-transferring enzyme acts on the saccharidehaving an α-isomaltosyl residue at the non-reducing end, and thenintermolecularly transfers the residue to the hydroxyl group at C-3position of a glucose residue of other saccharide having anα-isomaltosyl residue at the non-reducing end and forms a saccharidehaving an α-isomaltosyl-1,3-isomaltosyl residue at the non-reducing end;

[0102] (3) The α-isomaltosyl-transferring enzyme acts on a saccharidehaving an α-isomaltosyl-1,3-isomaltosyl residue at the non-reducing endto release the α-isomaltosyl-1,3-isomaltosyl residue from thesaccharide, and intramolecularly cyclizes the residue intocyclotetrasaccharide; and

[0103] (4) Through the steps (1) to (3), cyclotetrasaccharide is formedfrom a remaining saccharide which is formed by releasing theα-isomaltosyl-1,3-isomaltosyl residue in the step (3), and the yield ofcyclotetrasaccharide is highly increased by sequencially repeating thesteps (1) to (3).

[0104] As explained above, it can be estimated that, when used incombination, the polypeptide of the present invention andα-isomaltosyl-transferring enzyme act on their substrates repeatedly toincrease the yield of cyclotetrasaccharide.

[0105] During the cyclotetrasaccharide-forming reaction, optionally,other sacchride-transferring enzyme(s) can be advantageously used incombination to improve the yield of cyclotetrasaccharide; when two typesof enzymes, i.e., the polypeptide of the present invention andα-isomaltosy-transferring enzyme are allowed to act, for example, on anabout 15% solution of partial starch hydrolyzate, cyclotetrasaccharideis produced in a yield of about 55%, while the use of three types ofenzymes, i.e., the polypeptide of the present invention,α-isomaltosyl-transferring enzyme, and cyclomaltodextringlucanotransferase, under the same condition described above, increasesthe maximum yield of cyclotetrasaccharide by about 5-10% to an improvedyield of about 60-65%.

[0106] The saccharide solutions obtained by the above reaction can beused intact as solutions comprising cyclotetrasaccharide or saccharidecompositions of the same. In general, however, they can be purifiedbefore use by appropriate purification procedures. Conventionalprocedures can be appropriately selected as the purification procedures.For example, one or more of the following purification procedures can beused alone or in combination: Decoloration or purification withactivated charcoal, desalting by ion-exchange resins in a H— or OH-form,chromatographies such as shin-layer chromatography, high-performanceliquid chromatography, ion-exchange column chromatography, activatedcharcoal column chromatography, and silica gel column chromatography,separation using organic solvents such as alcohols and acetone, membraneseparation using adequate separability, hydrolysis of remainingsaccharides using amylases including α-amylase, β-amylase, glucoamylase(EC 3.2.1.3), and α-glucosidase (EC 3.2.1.20), and hydrolysis andremoval of the remaining saccharides by fermentation with yeast or byalkaline treatment. Particularly, ion-exchange column chromatography ispreferably used as an industrial scale production method; columnchromatography using strong-acid cation exchange resin as disclosed, forexample, in Japanese Patent Kokai Nos. 23,799/83 and 72,598/83. Usingthe column chromatography, the contaminating saccharides can be removedto advantageously produce cyclotetrasaccharide with an improved contentof the objective saccharide or saccharide compositions comprising thesame. In this case, any one of fixed-bed, moving bed, and semi-movingbed methods can be appropriately used.

[0107] The resulting cyclotetrasaccharide or saccharide compositionscomprising the same can be concentrated into syrup products, andoptionally they can be further dried into amorphous powdery products.

[0108] To produce cyclotetrasaccharide crystals, for example, highcyclotetrasaccharide content solutions, having a concentration of about30-90% and a purity of about 50% or higher of cyclotetrasaccharide, areplaced into a crystallizer optionally in the presence of an organicsolvent, and then gradually cooled while stirring in the presence of0.1-20%, d.s.b., of a seed crystal to the cyclotetrasaccharide at atemperature of 95° C. or lower, preferably, 10-90° C., to obtainmassecuites. The methods to produce cyclotetrasaccharide crystals andsaccharide comprising the same from the massecuites include, forexample, conventional methods such as block pulverization, fluidizedgranulation, and spray drying methods. Separation can be optionallyselected as the method to produce cyclotetrasaccharide crystals withmolasses.

[0109] The resulting cyclotetrasaccharide is a stable, high-quality, lowsweetness, non-reducing white powder or syrup, and is almost free ofbrowning, smelling, and deterioration of materials even when mixed orprocessed therewith: The materials are particularly, for example, aminoacid-containing substances such as amino acids, oligopeptides, andproteins. Since cyclotetrasaccharide has a clathrating ability, iteffectively inhibits the dispersion and quality deterioration offlavorful components and effective ingredients, and stably retains them.For such a purpose, if necessary, the combinational use ofcyclotetrasaccharide and other cyclic saccharide(s) such ascyclodextrins, branched-cyclodextrins, cyclodexrans, and cyclofructanscan be advantageously used to improve the level of the clathratingability of cyclotetrasaccharide. The above cyclic saccharides such ascyclodextrins should not be restricted to those with a high purity, andcyclic saccharides with a relatively-low purity such as partial starchhydrolyzates containing a large amount of maltodextrins and variouscyclodextrins can be advantageously used.

[0110] Since cyclotetrasaccharide, which is obtainable by using thepolypeptide of the present invention, is not substantially hydrolyzed byamylases and α-glucosidases, it is free of assimilation by the body whenorally administrated. Also, the saccharide is not substantiallyassimilated by intestinal bacteria, and therefore it can be used as anextremely-low caloric water-soluble dietary fiber. Cyclotetrasaccharidecan be also used as a sweetener substantially free from causing dentalcaries because it is scarcely assimilated by dental caries-inducingbacteria. The saccharide prevents the adhesion and solidification.Cyclotetrasaccharide per se is a nontoxic, harmless, safe and stablenatural sweetener. In the case of crystalline cyclotetrasaccharide, itcan be advantageously used for tablets and sugar-coated tablets incombination with binders such as pullulan, hydroxyethyl-starch, andpolyvinylpyrrolidone. Furthermore, cyclotetrasaccharide has usefulproperties of osmosis-controlling ability, filler-imparting ability,gloss-imparting ability, moisture-retaining ability, viscosity,crystallization-preventing ability for other saccharides, insubstantialfermentability, etc.

[0111] Thus, cyclotetrasaccharide and the saccharide compositionscomprising the same of the present invention can be arbitrary used as asweetener, taste-improving agent, quality-improving agent, stabilizer,preventive of discoloration, filler, etc., in a variety of compositionssuch as food products, tobaccos, cigarettes, feeds, pet foods,cosmetics, and pharmaceuticals.

[0112] Cyclotetrasaccharide and the saccharide compositions comprisingthe same of the present invention can be used intact as sweeteners. Ifnecessary, they can be advantageously used in combination with othersweeteners, for example, powdery syrup, glucose, fructose, lactose,isomerized sugar, sucrose, maltose, trehalose (α,α-trehalose,α,β-trehalose, and β,β-trehalose), honey, maple sugar, sorbitol,maltitol, dihydrochalcone, stevioside, α-glycosyl stevioside, sweetenerof Momordica grosvenori, glycyrrhizin, thaumatin, L-aspartylL-phenylalanine methyl ester, saccharine, acesulfame K, sucralose,glycine and alanine; and fillers such as dextrin, starch, and lactose.Particularly, cyclotetrasaccharide and the saccharide compositionscomprising the same can be suitably used as a low caloric sweetener,dietary sweetener, or the like in combination with one or morelow-caloric sweeteners such as erythritol, xylitol, and maltitol; and/orone or more sweeteners with a relatively-high sweetening power such asα-glycosyl stevioside, thaumatin, L-aspartyl L-phenylalanine methylester, saccharine, acesulfame K, and sucralose.

[0113] Cyclotetrasaccharide and the saccharide compositions comprisingthe same of the present invention can be arbitrarily used intact or, ifnecessary, after mixing with fillers, excipients, binders, etc., andthem formed into products with different shapes such as granules,spheres, sticks, plates, cubes, and tablets.

[0114] Cyclotetrasaccharide and the saccharide compositions comprisingthe same of the present invention well harmonize with other tastablematerials having sour-, salty-, bitter-, astringent-, delicious-, andbitter-taste; and have a high acid- and heat-tolerance. Thus, they canbe favorably used to sweeten and/or improve the taste, and quality offood products in general, for example, a soy sauce, powdered soy sauce,miso, “funmatsu-miso” (a powdered miso), “moromi” (a refined sake),“hishio” (a refined soy sauce), “furikake” (a seasoned fishmeal),mayonnaise, dressing, vinegar, “sanbai-zu” (a sauce of sugar, soy sauceand vinegar), “funmatsu-sushi-zu” (powdered vinegar for sushi),“chuka-no-moto” (an instant mix for Chinese dish), “tentsuyu” (a saucefor Japanese deep fat fried food), “mentsuyu” (a sauce for Japanesevermicelli), sauce, catsup, “yakiniku-no-tare” ( a sauce for Japanesegrilled meat), curry roux, instant stew mix, instant soup mix,“dashi-no-moto” (an instant stock mix), mixed seasoning, “mirin” (asweet sake), “shin-mirin” (a synthetic mirin), table sugar, and coffeesugar. Also, cyclotetrasaccharide and the saccharide compositionscomprising the same can be arbitrarily used to sweeten and improve thetaste, flavor, and quality of “wagashi” (Japanese cakes) such as“senbei” (a rice cracker), “arare” (a rice cake cube), “okoshi” (amillet and rice cake), “gyuhi” (a starch paste), “mochi” (a rise paste)and the like, “manju” (a bun with a bean-jam), “uiro” (a sweet ricejelly), “an” (a bean-jam) and the like, “yokan” (a sweet jelly ofbeans), “mizu-yokan” (a soft azuki-bean jelly), “kingyoku” (a kind ofyokan), jelly, pao de Castella, and “amedama” (a Japanese toffee);Western confectioneries such as a bun, biscuit, cracker, cookie, pie,pudding, butter cream, custardcream, cream puff, waffle, sponge cake,doughnut, chocolate, chewing gum, caramel, nougat, and candy; frozendesserts such as an ice cream and sherbet; syrups such as a“kajitsu-no-syrup-zuke” (a preserved fruit) and “korimitsu” (a sugarsyrup for shaved ice); pastes such as a flour paste, peanut paste, andfruit paste; processed fruits and vegetables such as a jam, marmalade,“syrup-zuke” (fruit pickles), and “toka” (conserves); pickles andpickled products such as a “fukujin-zuke” (red colored radish pickles),“bettara-zuke” (a kind of whole fresh radish pickles), “senmai-zuke” (akind of sliced fresh radish pickles), and “rakkyo-zuke” (pickledshallots); premix for pickles and pickled products such as a“takuan-zuke-no-moto” (a premix for pickled radish), and“hakusai-zuke-no-moto” (a premix for fresh white rape pickles); meatproducts such as a ham and sausage; products of fish meat such as a fishham, fish sausage, “kamaboko” (a steamed fish paste), “chikuwa” (a kindof fish paste), and “tenpura” (a Japanease deep-fat fried fish paste);“chinmi” (relish) such as a “uni-no-shiokara” (salted guts of urchin),“ika-no-shiokara” (salted guts of squid), “su-konbu” (processed tangle),“saki-surume” (dried squid strips), “fugu-no-mirin-boshi” (a driedmirin-seasoned swellfish), seasoned fish flour such as of Pacific cod,sea bream, shrimp, etc.; “tsukudani” (foods boiled down in soy sauce)such as those of laver, ediblewildplants, dried squid, small fish, andshellfish; daily dishes such as a “nimame” (cooked beans), potato salad,and “konbu-maki” (a tangle roll); milk products; canned and bottledproducts such as those of meat, fish meat, fruit, and vegetable;alcoholic beverages such as a synthetic sake, fermented liquor, fruitliquor, and sake; soft drinks such as a coffee, cocoa, juice, carbonatedbeverage, sour milk beverage, and beverage containing a lactic acidbacterium; instant food products such as instant pudding mix, instanthot cake mix, instant juice, instant coffee, “sokuseki-shiruko” (aninstant mix of azuki-bean soup with rice cake), and instant soup mix;and other foods and beverages such as solid foods for babies, foods fortherapy, drinks, beverage containing amino acids, peptide foods, andfrozen foods.

[0115] Cyclotetrasaccharide and the saccharide compositions comprisingthe same can be arbitrarily used to improve the taste preference offeeds and pet foods for animals and pets such as domestic animals,poultry, honey bees, silk warms, and fishes; and also they can bearbitrarily used as a sweetener and taste-improving agent, taste-curingagent, quality-improving agent, and stabilizer in other products in apaste or liquid form such as tobacco, cigarette, tooth paste, lipstick,rouge, lip cream, internal liquid medicine, tablet, troche, cod-liveroil in the form of drop, oral refrigerant, cachou, gargle, cosmetic andpharmaceutical. When used as a quality-improving agent or stabilizer,cyclotetrasaccharide and the saccharide compositions comprising the samecan be arbitrarily used in biologically active substances susceptible tolose their effective ingredients and activities, as well as in healthfoods, cosmetics, and pharmaceuticals containing the biologically activesubstances. Example of such biologically active substances are liquidpreparations containing cytokines such as α-, β-, and γ-interferons,tumor necrosis factor-α (TNF-α), tumor necrosis factor-β (TNF-β),macropharge migration inhibitory factor, colony-stimulating factor,transfer factor, and interleukin 2; liquid preparations containinghormones such as insulin, growth hormone, prolactin, erythropoietin, andfollicle-stimulating hormone; biological preparations such as BCGvaccine, Japanese encephalitis vaccine, measles vaccine, live poliovaccine, small pox vaccine, tetanus toxoid, Trimeresurus antitoxin, andhuman immunoglobulin; antibiotics such as penicillin, erythromycin,chloramphenicol, tetracycline, streptmycin, and kanamycin sulfate;vitamins such as thiamin, ribofravin, L-ascorbic acid, cod liver oil,carotenoide, ergosterol, tocopherol; solution of enzymes such as lipase,esterase, urokinase, protease, β-amylase, isoamylase, glucanase, andlactase; extracts such as ginseng extract, turtle extract, chlorellaextract, aloe extract, and propolis extract; and royal jelly. By usingcyclotetrasaccharide and the saccharide compositions comprising thesame, the above biologically active substances and other paste of livingmicroorganisms such as virus, lactic acid bacteria, and yeast can bearbitrary prepared into health foods and pharmaceuticals in a liquid,paste, or solid form, which have a satisfactorily-high stability andquality with less fear of losing or inactivating their effectiveingredients and activities.

[0116] The methods for incorporating cyclotetrasaccharide or thesaccharide composition comprising the same into the aforesaidcompositions are those which can incorporate cyclotetrasaccharide andthe saccharide compositions into a variety of compositions beforecompletion of their processing, and which can be appropriately selectedamong the following conventional methods; mixing, kneading, dissolving,melting, soaking, penetrating, dispersing, applying, coating, spraying,injecting, crystallizing, and solidifying. The amount of thecyclotetrasaccharide or the saccharide compositions comprising the sameto be preferably incorporated into the final compositions is usually inan amount of 0.1% or higher, desirably, 1% or higher.

[0117] The following experiments explain the physicochemical propertiesof the polypeptides having an α-isomaltosylglucosaccharide-formingenzyme activity from Bacillus globisporus C11, Bacillus globisporus N75,and Arthrobacter globiformis A19, DNAs encoding the polypeptide havingan α-isomaltosylglucosaccharide-forming enzyme activity, and thepreparation methods of recombinant polypeptides having anα-isomaltosylglucosaccharide-forming enzyme activity.

[0118] Experiment 1

[0119] Preparation of a Polypeptide having anα-Isomaltosylglucosaccharide-Forming Enzyme Activity from Bacillusglobisporus C11

[0120] Experiment 1-1

[0121] Preparation of α-Isomaltosylglucosaccharide-Forming Enzyme

[0122] A liquid culture medium consisting 4% (w/v) of “PINE-DEX #4”, apartial starch hydrolyzate, 1.8% (w/v) of “ASAHIMEAST”, a yeast extract,0.1% (w/v) of dipotassium phosphate, 0.06% (w/v) of sodium phosphatedodeca-hydrate, 0.05% (w/v) of magnesium sulfate hepta-hydrate, andwater was placed in 500-ml Erlenmeyer flasks in a respective amount of100 ml, sterilized by autoclaving at 121° C. for 20 minutes, cooled andseeded with Bacillus globisporus C11 strain, followed by culturing underrotary-shaking conditions at 27° C. and 230 rpm for 48 hours for a seedculture. About 20 L of a fresh preparation of the same liquid culturemedium as used in the above seed culture were placed in a 30 Lfermentor, sterilized by heating, and then cooled to 27° C. andinoculated with 1% (v/v) of the seed culture, followed by culturing at27° C. and pH. 6.0 to 8.0 for 48 hours under aeration-agitationconditions. After the completion of the culture, about 0.55 unit/ml ofα-isomaltosylglucosaccharide-forming enzyme and about 1.8 units/ml ofα-isomaltosyl-transferring enzyme were detected in the resulting cultureby measuring enzyme activities. About 18 L of supernatant obtained bycentrifugation (10,000 rpm, 30 minutes) had about 0.51 units/ml ofα-isomaltosylglucosaccharide-forming enzyme activity, i.e., a totalactivity of about 9,180 units; and about 1.7 units/ml ofα-isomaltosyl-transferring enzyme activity, i.e., a total enzymaticactivity of about 30,400 units. Since both enzyme activities weredetected mainly in culture supernatant, it was revealed that theseenzymes were secretion enzymes secreted in the culture.

[0123] The two types of enzymatic activities described above weremeasured as following. The activity ofα-isomaltosylglucosaccharide-forming enzyme was measured by thefollowing assay: A substrate solution was prepared by dissolvingmaltotriose in 100 mM acetate buffer (pH 6.0) to give a concentration of2% (w/v). A reaction mixture was prepared by mixing 0.5 ml of thesubstrate solution and 0.5 ml of an enzyme solution, and incubated at35° C. for 60 minutes. After stopping the reaction by boiling for 10minutes, the amount of maltose formed in the reaction mixture wasdetermined by high-performance liquid chromatography (HPLC). One unit ofα-isomaltosylglucosaccharide-forming activity was defined as the amountof the enzyme that forms one μmole of maltose per minute under the aboveconditions. HPLC was carried out using “SHODEX KS-801 column”, ShowaDenko K. K., Tokyo, Japan, at a column temperature of 60° C. and a flowrate of 0.5 ml/minutes of water, and using “RI-8012”, a differentialrefractometer commercialized by Tosoh Corporation, Tokyo, Japan.

[0124] The activity of α-isomaltosyl-transferring enzyme was measured bythe following assay: A substrate solution was prepared by dissolvingpanose in 100 mM acetate buffer (pH 6.0) to give a concentration of 2%(w/v). A reaction mixture was prepared by mixing 0.5 ml of the substratesolution and 0.5 ml of an enzyme solution, and incubated at 35° C. for30 minutes. After stopping the reaction by boiling for 10 minutes, theamount of glucose formed in the reaction mixture was determined by theglucose oxidase-peroxidase method. One unit ofα-isomaltosyl-transferring activity was defined as the amount of theenzyme that forms one μmole of glucose per minute under the aboveconditions.

[0125] Experiment 1-2

[0126] Preparation of Partially Purified Enzymes

[0127] About 18 L of the culture supernatant obtained in Experiment 1-1were salted out with 80% saturated ammonium sulfate solution and allowedto stand at 4° C. for 24 hours, and the formed precipitates werecollected by centrifugation (10,000 rpm, 30 minutes), dissolved in 10 mMsodium phosphate buffer (pH 7.5), and dialyzed against the same bufferto obtain about 416 ml of a crude enzyme solution. The crude enzymesolution had about 8,440 units of α-isomaltosylglucosaccharide-formingenzyme and about 28,000 units of α-isomaltosyl-transferring enzyme. Thecrude enzyme solution was subjected to ion-exchange columnchromatography using “SEPABEADS FP-DA13” gel, an ion-exchange resincommercialized by Mitsubishi Chemical Industries, Ltd., Tokyo, Japan.Both α-isomaltosylglucosaccharide-forming enzyme andα-isomaltosyl-transferring enzyme were eluted as non-adsorbed fractionswithout adsorbing on “SEPABEADS FP-DA13” gel. The non-adsorbed fractionwas collected and dialyzed against 10 mM sodium phosphate buffer (pH7.0) with 1 M ammonium sulfate. The dialyzed solution was centrifuged toremove impurities, and subjected to affinity column chromatography using500 ml of “SEPHACRYL HR S-200” gel, a gel commercialized by AmershamBiosciences K. K., Tokyo, Japan (old name, Amersham Pharmacia Biotech).Enzymatically active components adsorbed on “SEPHACRY HR S-200” gel and,when sequentially eluted with a linear gradient decreasing from 1 M to 0M of ammonium sulfate and a linear gradient increasing from 0 mM to 100mM of maltotetraose, the α-isomaltosyl-transferring enzyme and theα-isomaltosylglucosaccharide-forming enzyme were separately eluted,i.e., the former was eluted with a linear gradient of ammonium sulfateat about 0.3 M and the latter was eluted with a linear gradient ofmaltotetraose at about 30 mM. Thus, fractions with theα-isomaltosylglucosaccharide-forming enzyme activity and those with theα-isomaltosyl-transferring enzyme activity were separately collected aspartially purified preparations of α-isomaltosylglucosaccharide-formingenzyme and α-isomaltosyl-transferring enzyme. Further, these enzymepreparations were purified separately.

[0128] Experiment 1-3

[0129] Purification of a Polypeptide having anα-Isomaltosylglucosaccharide-Forming Enzyme Activity

[0130] The partially purified enzyme preparation havingα-isomaltosylglucosaccharide-forming enzyme activity, obtained inExperiment 1-2, was dialyzed against 10 mM sodium phosphate buffer (pH7.0) with 1 M ammonium sulfate. The dialyzed solution was centrifuged toremove impurities, and subjected to hydrophobic column chromatographyusing 350 ml of “BUTYL-TOYOPEARL 650M” gel, a hydrophobic gelcommercialized by Tosoh Corporation, Tokyo, Japan. The enzyme wasadsorbed on “BUTYL-TOYOPEARL 650M” gel and, when eluted with a lineargradient decreasing from 1 M to 0M of ammonium sulfate, the enzymaticactivity was eluted with a linear gradient of ammonium sulfate at about0.3 M, and fractions with the enzyme activity was collected. Thecollected solution was dialyzed against 10 mM sodium phosphate buffer(pH 7.0) with 1 M ammonium sulfate, and the dialyzed solution wascentrifuged to remove impurities, and purified by affinitychromatography using “SEPHACRYL HR S-200” gel. The amount of enzymeactivity, specific activity and yield of theα-isomaltosylglucosaccharide-forming enzyme in each purification stepare shown in Table 1. TABLE 1 Enzyme* Specific activity activity ofenzyme* Yield Purification step (unit) (unit/mg protein) (%) Culturesupernatant 9,180 0.14 100 Dialyzed solution after 8,440 0.60 91.9salting out with ammonium sulfate Elute from ion-exchange 6,620 1.0872.1 column chromatography Elute from affinity column 4,130 8.83 45.0chromatography Elute from hydrophobic 3,310 11.0 36.1 columnchromatography Elute from affinity column 2,000 13.4 21.8 chromatography

[0131] The finally purified α-isomaltosylglucosaccharide-forming enzymepolypeptide specimen was assayed for purity on gel electrophoresis usinga 7.5% (w/v) polyacrylamide gel and detected on the gel as a singleprotein band, i.e., a high purity specimen.

[0132] Experiment 1-4

[0133] Purification of a Polypeptide having anα-Isomaltosyl-Transferring Enzyme Activity

[0134] The partially purified enzyme preparation havingα-isomaltosyl-transferring enzyme activity, obtained in Experiment 1-2,was dialyzed against 10 mM sodium phosphate buffer (pH 7.0) with 1 Mammonium sulfate. The dialyzed solution was centrifuged to removeimpurities, and subjected to hydrophobic column chromatography using 350ml of “BUTYL-TOYOPEARL 650M” gel, a hydrophobic gel commercialized byTosoh Corporation, Tokyo, Japan. The enzyme adsorbed on “BUTYL-TOYOPEARL650M” gel and, when eluted with a linear gradient decreasing from 1 M to0M of ammonium sulfate, the enzymatically active fractions were elutedwith a linear gradient of ammonium sulfate at about 0.3 M, and fractionswith the enzyme activity was collected. The collected solution wasdialyzed against 10 mM sodium phosphate buffer (pH 7.0) with 1 Mammonium sulfate, and the dialyzed solution was centrifuged to removeimpurities, and purified by affinity chromatography using “SEPHACRYL HRS-200” gel. The amount of enzyme activity, specific activity and yieldof the α-isomaltosyl-transferring enzyme in each purification step arein Table 2. Enzyme* Specific activity activity of enzyme* YieldPurification step (unit) (unit/mg protein) (%) Culture supernatant30,400 0.45 100 Dialyzed solution after 28,000 1.98 92.1 salting outwith ammonium sulfate Elute from ion-exchange 21,800 3.56 71.7 columnchromatography Elute from affinity column 13,700 21.9 45.1chromatography Elute from hydrophobic 10,300 23.4 33.9 columnchromatography Elute from affinity column 5,510 29.6 18.1 chromatography

[0135] The finally purified α-isomaltosyl-transferring enzyme specimenwas assayed for purify on gel electrophoresis using a 7.5% (w/v)polyacrylamide gel and detected on the gel as a single protein band,i.e., a high purity polypeptide.

[0136] Experiment 2

[0137] Preparation of a Polypeptide having anα-Isomaltosylglucosaccharide-Forming Enzyme Activity from Bacillusglobisporus N75

[0138] Experiment 2-1

[0139] Preparation of α-Isomaltosylglucosaccharide-Forming Enzyme

[0140] A liquid culture medium consisting 4% (w/v) of “PINE-DEX #4”, apartial starch hydrolyzate, 1.8% (w/v) of “ASAHIMEAST”, a yeast extract,0.1% (w/v) of dipotassium phosphate, 0.06% (w/v) of sodium phosphatedodeca-hydrate, 0.05% (w/v) of magnesium sulfate hepta-hydrate, andwater was placed in 500-ml Erlenmeyer flasks in a respective amount of100 ml, sterilized by autoclaving at 121° C. for 20 minutes, cooled andseeded with Bacillus globisporus N75, followed by culturing underrotary-shaking conditions at 27° C. and 230 rpm for 48 hours for a seedculture. About 20 L of a fresh preparation of the same liquid culturemedium as used in the above seed culture were placed in a 30 Lfermentor, sterilized by heating, and then cooled to 27° C. andinoculated with 1% (v/v) of the seed culture, followed by culturing at27° C. and pH 6.0 to 8.0 for 48 hours under aeration-agitationconditions. After the completion of the culture, about 0.34 unit/ml ofα-isomaltosylglucosaccharide-forming enzyme and about 1.1 units/ml ofα-isomaltosyl-transferring enzyme were detected in the resulting cultureby measuring enzyme activities. About 18 L of supernatant obtained bycentrifugation (10,000 rpm, 30 minutes) had about 0.33 units/ml ofα-isomaltosylglucosaccharide-forming enzyme activity, i.e., a totalactivity of about 5,940 units; and about 1.1 units/ml ofα-isomaltosyl-transferring enzyme activity, i.e., a total enzymaticactivity of about 19,800 units. Since both enzyme activities weredetected mainly in culture supernatant, it was revealed that theseenzymes were secretion enzymes secreted in the culture.

[0141] Experiment 2-2

[0142] Preparation of Partially Purified Enzymes

[0143] About 18 L of the culture supernatant obtained in Experiment 2-1was salted out with 80% saturated ammonium sulfate solution and allowedto stand at 4° C. for 24 hours, and the formed precipitates werecollected by centrifugation (10,000 rpm, 30 minutes), dissolved in 10 mMTris-HCl buffer (pH 8.3), and dialyzed against the same buffer to obtainabout 450 ml of crude enzyme solution. The crude enzyme solution hadabout 4,710 units of α-isomaltosylglucosaccharide-forming enzymeactivity and about 15,700 units of α-isomaltosyl-transferring enzymeactivity. The crude enzyme solution was subjected to ion-exchange columnchromatography using “SEPABEADS FP-DA13” gel, disclosed in Experiment1-1. α-Isomaltosylglucosaccharide-forming enzyme was adsorbed on“SEPABEADS FP-DA13” gel, and α-isomaltosyl-transferring enzyme waseluted as non-adsorbed fraction without adsorbing on “SEPABEADS FP-DA13”gel. Subsequently, α-isomaltosylglucosaccharide-forming enzyme waseluted with a linear gradient of increasing from 0 M to 1 M of sodiumchloride, where the enzyme was eluted with the linear gradient of sodiumchloride at a concentration of about 0.25 M. Therefore, fractions withα-isomaltosylglucosaccharide-forming enzyme and withα-isomaltosyl-transferring enzyme were separately collected as partiallypurified enzyme preparation having α-isomaltosylglucosaccharide-formingenzyme activity and that having α-isomaltosyl-transferring enzymeactivity, respectively. Further, these enzyme preparations wereseparately purified.

[0144] Experiment 2-3

[0145] Purification of a Polypeptide havingα-Isomaltosylglucosaccharide-Forming Enzyme Activity

[0146] The partially purified enzyme preparation havingα-isomaltosylglucosaccharide-forming enzyme activity, obtained inExperiment 2-2, was dialyzed against 10 mM sodium phosphate buffer (pH7.0) with 1 M ammonium sulfate. The dialyzed solution was centrifuged toremove impurities, and subjected to affinity column chromatography using500 ml of “SEPHACRYL HR S-200” gel, a gel commercialized by AmershamBiosciences K. K., Tokyo, Japan (old name, Amersham Pharmacia Biotech).The enzyme was adsorbed on “SEPHACRYL HR S-200” gel and, whensequentially eluted with a linear gradient decreasing from 1 M to 0 M ofammonium sulfate and a linear gradient increasing from 0 mM to 100 mM ofmaltotetraose, the enzymatic activity was eluted with a linear gradientof maltotetraose at about 30 mM, and fractions with the enzyme activitywas collected. The collected solution was dialyzed against 10 mM sodiumphosphate buffer (pH 7.0) with 1 M ammonium sulfate, and the dialyzedsolution was centrifuged to remove impurities, and purified byhydrophobic column chromatography using 350 ml of “BUTYL-TOYOPEARL 650M”gel, a hydrophobic gel commercialized by Tosoh Corporation, Tokyo,Japan. The enzyme was adsorbed on “BUTYL-TOYOPEARL 650M” gel and, wheneluted with a linear gradient decreasing from 1 M to 0 M of ammoniumsulfate, the enzymatic activity was eluted with a linear gradient ofammonium sulfate at about 0.3 M, and fractions with the enzyme activitywas collected. The collected solution was dialyzed against 10 mM sodiumphosphate buffer (pH 7.0) with 1 M ammonium sulfate, and the dialyzedsolution was centrifuged to remove impurities, and purified by affinitychromatography using “SEPHACRLY HR S-200” gel. The amount of enzymeactivity, specific activity and yield of theα-isomaltosylglucosaccharide-forming enzyme in each purification stepare shown in Table 3. Enzyme* Specific activity activity of enzyme*Yield Purification step (unit) (unit/mg protein) (%) Culture supernatant5,940 0.10 100 Dialyzed solution after 4,710 0.19 79.3 salting out withammonium sulfate Elute from ion-exchange 3,200 2.12 53.9 columnchromatography Elute from affinity column 2,210 7.55 37.2 chromatographyElute from hydrophobic 1,720 10.1 29.0 column chromatography Elute fromaffinity column 1,320 12.5 22.2 chromatography

[0147] The finally purified α-isomaltosylglucosaccharide-forming enzymespecimen was assayed for purify on gel electrophoresis using a 7.5%(w/v) polyacrylamide gel and detected on the gel as a single proteinband, i.e., a high purity polypeptide.

[0148] Experiment 2-4

[0149] Purification of a Polypeptide having anα-Isomaltosyl-Transferring Enzyme Activity

[0150] The partially purified enzyme preparation havingα-isomaltosyl-transferring activity, obtained in Experiment 2-2, wasdialyzed against 10 mM sodium phosphate buffer (pH 7.0) with 1 Mammonium sulfate. The dialyzed solution was centrifuged to removeimpurities, and subjected to affinity column chromatography using 500 mlof “SEPHACRYL HR S-200” gel, a gel commercialized by AmershamBiosciences K. K., Tokyo, Japan (old name, Amersham Pharmacia Biotech).The enzyme was adsorbed on “SEPHACRYL HR S-200” gel and, when elutedwith a linear gradient decreasing from 1 M to 0M of ammonium sulfate,the enzymatic activity was eluted with a linear gradient of ammoniumsulfate at about 0.3 M, and fractions with the enzyme activity wascollected. The collected solution was dialyzed against 10 mM sodiumphosphate buffer (pH 7.0) with 1 M ammonium sulfate, and the dialyzedsolution was centrifuged to remove impurities, and purified byhydrophobic column chromatography using “BUTYL-TOYOPEARL 650M” gel, ahydrophobic gel commercialized by Tosoh Corporation, Tokyo, Japan. Thepolypeptide was adsorbed on “BUTYL-TOYOPEARL 650M” gel and, when elutedwith a linear gradient decreasing from 1 M to 0M of ammonium sulfate,the enzymatic activity was eluted with a linear gradient of ammoniumsulfate at about 0.3 M, and fractions with the enzyme activity wascollected. The collected solution was dialyzed against 10 mM Tris-HClbuffer (pH 8.0), and the dialyzed solution was centrifuged to removeimpurities, and purified by ion-exchange column chromatography using 380ml of “SUPER Q-TOYOPEARL 650C” gel, an ion-exchange gel commercializedby Tosoh Corporation, Tokyo, Japan. The enzyme was eluted asnon-adsorbed fraction without adsorbing on “SUPER Q-TOYOPEARL 650C” gel.The purified polypeptide specimen having α-isomaltosyl-transferringenzyme activity was obtained by collecting the fractions. The amount ofenzyme activity, specific activity and yield of theα-isomaltosyl-transferring enzyme in each purification step are shown inTable 4. TABLE 4 Enzyme* Specific activity activity of enzyme* YieldPurification step (unit) (unit/mg protein) (%) Culture supernatant19,000 0.33 100 Dialyzed solution after 15,700 0.64 82.6 salting outwith ammonium sulfate Elute from ion-exchange 12,400 3.56 65.3 columnchromatography Elute from affinity column 8,320 11.7 43.8 chromatographyElute from hydrophobic 4,830 15.2 25.4 column chromatography Elute fromion-exchange 3,850 22.6 20.3 column chromatography

[0151] The finally purified α-isomaltosyl-transferring enzyme 5 specimenwas assayed for purify on gel electrophoresis using a 7.5% (w/v)polyacrylamide gel and detected on the gel as a single protein band,i.e., a high purity polypeptide.

[0152] Experiment 3

[0153] Preparation of a Polypeptide having anα-Isomaltosylglucosaccharide-Forming Enzyme Activity from Arthrobacterglobiformis A19

[0154] Experiment 3-1

[0155] Preparation of α-Isomaltosylglucosaccharide-Forming Enzyme

[0156] A liquid culture medium consisting 4% (w/v) of “PINE-DEX #4”, apartial starch hydrolyzate, 1.8% (w/v) of “ASAHIMEAST”, a yeast extract,0.1% (w/v) of dipotassium phosphate, 0.06% (w/v) of sodium phosphatedodeca-hydrate, 0.05% (w/v) of magnesium sulfate hepta-hydrate, andwater was placed in 500-ml Erlenmeyer flasks in a respective amount of100 ml, sterilized by autoclaving at 121° C. for 20 minutes, cooled andseeded with Arthrobacter globiformis A19, followed by culturing underrotary-shaking conditions at 27° C. and 230 rpm for 48 hours for a seedculture. About 20 L of a fresh preparation of the same liquid culturemedium as used in the above seed culture were placed in a 30 Lfermentor, sterilized by heating, and then cooled to 27° C. andinoculated with 1% (v/v) of the seed culture, followed by culturing at27° C. and pH 6.0 to 9.0 for 48 hours under aeration-agitationconditions. After the completion of the culture, about 1.1 unit/ml ofα-isomaltosylglucosaccharide-forming enzyme and about 1.7 units/ml ofα-isomaltosyl-transferring enzyme were detected in the resulting cultureby measuring enzyme activities. About 18 L of supernatant obtained bycentrifugation (10,000 rpm, 30 minutes) had about 1.06 units/ml ofα-isomaltosylglucosaccharide-forming enzyme activity, i.e., a totalactivity of about 19,100 units; and about 1.6 units/ml ofα-isomaltosyl-transferring enzyme activity, i.e., a total enzymaticactivity of about 28,800 units. Since both enzyme activities weredetected mainly in culture supernatant, it was revealed that theseenzymes were secretion enzymes secreted in the culture. Except for using100 mM Glycine-NaOH buffer (pH 8.4) as the buffer to dissolve thesubstrate, the activity of α-isomaltosylglucosaccharide-forming enzymefrom Arthrobacter globiformis A19 was measured according to the methoddescribed in Experiment 1

[0157] Experiment 3-2

[0158] Preparation of Partially Purified Enzymes

[0159] About 18 L of the culture supernatant obtained in Experiment 3-1was salted out with 60% saturated ammonium sulfate solution and allowedto stand at 4° C. for 24 hours, and the formed precipitates werecollected by centrifugation (10,000 rpm, 30 minutes), dissolved in 10 mMTris-HCl buffer (pH 7.0), and dialyzed against the same buffer to obtainabout 850 ml of crude enzyme solution. The crude enzyme solution hadabout 8,210 units of α-isomaltosylglucosaccharide-forming enzymeactivity and about 15,700 units of α-isomaltosyl-transferring enzymeactivity. The crude enzyme solution was subjected to ion-exchange columnchromatography using 380 ml of “DEAE-TOYOPEARL 650S” gel, anion-exchange gel commercialized by Tosoh Corporation, Tokyo, Japan. Bothenzymes were adsorbed on “DEAE-TOYOPEARL 650S” gel and eluted with alinear gradient of increasing from 0 M to 1 M of sodium chloride, whereα-isomaltosylglucosaccharide-forming enzyme andα-isomaltosyl-transferring enzyme were eluted with the linear gradientof sodium chloride at concentrations of about 0.2 M and about 0.3 M,respectively. Therefore, fractions withα-isomaltosylglucosaccharide-forming enzyme and withα-isomaltosyl-transferring enzyme were separately collected as partiallypurified enzyme preparation having α-isomaltosylglucosaccharide-formingenzyme activity and that having α-isomaltosyl-transferring enzymeactivity, respectively. Further, these enzyme preparations wereseparately purified.

[0160] Experiment 3-3

[0161] Purification of a Polypeptide havingα-Isomaltosylglucosaccharide-Forming Enzyme Activity

[0162] The partially purified enzyme preparation havingα-isomaltosylglucosaccharide-forming enzyme activity, obtained inExperiment 3-2, was dialyzed against 10 mM sodium phosphate buffer (pH7.0) with 1 M ammonium sulfate. The dialyzed solution was centrifuged toremove impurities, and subjected to affinity column chromatography using500 ml of “SEPHACRYL HR S-200” gel, a gel commercialized by AmershamBiosciences K. K., Tokyo, Japan (old name, Amersham Pharmacia Biotech).The enzyme was adsorbed on “SEPHACRYL HR S-200” gel and, when elutedwith a linear gradient decreasing from 1 M to 0 M of ammonium sulfate,the enzymatic activity was eluted with a linear gradient of ammoniumsulfate at about 0.2 M, and fractions with the enzyme activity wascollected as purified enzyme preparation. The amount of enzyme activity,specific activity and yield of the α-isomaltosylglucosaccharide-formingenzyme in each purification step are shown in Table 5. TABLE 5 Enzyme*Specific activity activity of enzyme* Yield Purification step (unit)(unit/mg protein) (%) Culture supernatant 19,100 0.11 100 Dialyzedsolution after 8,210 0.48 43.0 salting out with ammonium sulfate Elutefrom ion-exchange 6,890 4.18 36.1 column chromatography Elute fromaffinity column 5,220 35.1 27.3 chromatography

[0163] The finally purified α-isomaltosylglucosaccharide-forming enzymespecimen was assayed for purity on gel electrophoresis using a 7.5%(w/v) polyacrylamide gel and detected on the gel as a single proteinband, i.e., a high purity polypeptide.

[0164] Experiment 3-4

[0165] Partial Purification of a Polypeptide having anα-Isomaltosyl-Transferring Enzyme Activity

[0166] The partially purified enzyme preparation havingα-isomaltosyl-transferring activity, obtained in Experiment 3-2, wasdialyzed against 10 mM sodium phosphate buffer (pH 7.0) with 1 Mammonium sulfate. The dialyzed solution was centrifuged to removeimpurities, and subjected to affinity column chromatography using 500 mlof “SEPHACRYL HR S-200” gel, a gel commercialized by AmershamBiosciences K. K., Tokyo, Japan (old name, Amersham Pharmacia Biotech).The enzyme was adsorbed on “SEPHACRYL HR S-200” gel and, when elutedwith a linear gradient decreasing from 1 M to 0 M of ammonium sulfate,the enzymatic activity was eluted with a linear gradient of ammoniumsulfate at about 0 M, and fractions with the enzyme activity wascollected as partially purified enzyme preparation. The amount of enzymeactivity, specific activity and yield of the α-isomaltosyl-transferringenzyme in each purification step are shown in Table 6. TABLE 6 Enzyme*Specific activity activity of enzyme* Yield Purification step (unit)(unit/mg protein) (%) Culture supernatant 28,800 0.18 100 Dialyzedsolution after 15,700 0.97 54.5 salting out with ammonium sulfate Elutefrom ion-exchange 7,130 4.01 24.8 column chromatography Elute fromaffinity column 1,800 11.9 6.3 chromatography

[0167] The partially purified α-isomaltosyl-transferring enzyme specimenwas assayed for purity on gel electrophoresis using a 7.5% (w/v)polyacrylamide gel and detected on the gel as one main and three minorprotein bands.

[0168] Experiment 4-1

[0169] Action on Saccharides

[0170] It was tested whether saccharides can be used as substrates forthe polypeptide of α-isomaltosylglucosaccharide-forming enzyme of thepresent invention. For the purpose, a solution of maltose, maltotriose,maltotetraose, maltopentaose, maltohexaose, maltoheptaose, isomaltose,isomaltotriose, panose, isopanose, trehalose, kojibiose, nigerose,neotrehalose, cellobiose, gentibiose, maltitol, maltotriitol, lactose,sucrose, erlose, selaginose, maltosyl glucoside, or isomaltosylglucoside was prepared.

[0171] To each of the above solutions was added two units/g substrate ofa purified polypeptide specimen of α-isomaltosylglucosaccharide-formingenzyme from either Bacillus globisporus C11 obtained by the method inExperiment 1-3, Bacillus globisporus N75 obtained by the method inExperiment 2-3, or Arthrobacter globiformis A19 obtained by the methodin Experiment 3-3, and the resulting each solution was adjusted to givea substrate concentration of 2% (w/v) and incubated at 30° C. and pH6.0, except for using pH 8.4 for the enzyme from Arthrobacterglobiformis A19, for 24 hours. In order to analyze the saccharides inthe reaction mixture before and after the enzymatic reaction, silica gelthin-layer chromatography (hereinafter, abbreviated as “TLC”) wascarried out. After two-times development using, as a developer, amixture solution of n-butanol, pyridine, and water (=6:4:1), and, as athin-layer plate, “KIESELGEL 60”, an aluminum plate (20×20 cm) for TLCcommercialized by Merck & Co., Inc., Rahway, USA, the saccharides in thereaction mixture were examined whether the enzyme acted or not by thecoloration of sugars by the sulfuric acid-methanol method. The resultsare shown in Table 7. TABLE 7 Enzyme action Substrate C11 enzyme N75enzyme A19 enzyme Maltose + + + Maltotriose ++ ++ ++ Maltotetraose ++++++ +++ Maltopentaose +++ +++ +++ Maltohexaose +++ +++ +++ Maltoheptaose+++ +++ +++ Isomaltose − − − Isomaltotriose − − − Panose − − − Isopanose++ ++ ++ Trehalose − − − Kojibiose + + + Nigerose + + +Neotrehalose + + + Cellobiose − − − Gentibiose − − − Maltitol − − −Maltotriitol + + + Lactose − − − Sucrose − − − Erlose + + + Selaginose −− − Maltosyl glucoside ++ ++ ++ Isomaltosyl glucoside − − − # of otherreaction product, and it showed a substantial disappearance of thesubstrate spot and the formation of other reaction product,respectively.

[0172] As evident from the Table 7, it was revealed that the polypeptidehaving α-isomaltosylglucosaccharide-forming enzyme activity well actedon saccharide having a glucose polymerization degree of 3 or higher andbearing a maltose structure at their non-reducing ends, among thesaccharides tested. It was also found that the polypeptide slightlyacted on saccharides, having glucose polymerization degree of two, suchas maltose, kojibiose, nigerose, neotrehalose, maltotriitol, and erlose.

[0173] Experiment 4-2

[0174] Reaction Product from Maltooligosaccharide

[0175] To an aqueous solution containing one percent (w/v) of maltose,maltotriose, maltotetraose, or maltopentaose as a substrate was addedthe polypeptide having α-isomaltosylglucosaccgaride-forming enzymeactivity, obtained in Experiment 1-3, in an amount of two units/g-solidfor maltose and maltotriose, 0.2 unit/g-solid for maltotetraose, and 0.1unit/g-solid for maltopentaose, followed by incubation at 35° C. and pH6.0 for 8 hours. After stopping the enzymatic reaction by a 10-minutesincubation, sugar compositions in the reaction mixture were measured byHPLC using “YMC Pack ODS-AQ303”, a column commercialized by YMC Co.,Ltd., Tokyo, Japan, at a column temperature of 40° C. and a flow rate of0.5 ml/minutes of water, and using as a detector “RI-8012”, adifferential refractometer commercialized by Tosoh Corporation, Tokyo,Japan. The results are in Table 8. TABLE 8 Saccharide as ReactionSubstrate product Mlatose Maltotriose Maltotetraose MaltopentaoseGlucose 8.5 0.1 0.0 0.0 Maltose 78.0 17.9 0.3 0.0 Maltotriose 0.8 45.322.7 1.9 Maltotetraose 0.0 1.8 35.1 19.2 Maltopentaose 0.0 0.0 3.5 34.4Maltohexaose 0.0 0.0 0.0 4.6 Isomaltose 0.5 0.0 0.0 0.0 Glucosyl- 8.21.2 0.0 0.0 maltose Glucosyl- 2.4 31.5 6.8 0.0 maltotriose X 0.0 2.130.0 11.4 Y 0.0 0.0 1.4 26.8 Z 0.0 0.0 0.0 1.7 Others 0.6 0.1 0.2 0.0

[0176] As evident from the results in Table 8, it was revealed that,after the action of the polypeptide havingα-isomaltosylglucosaccharide-forming enzyme activity, glucose andα-isomaltosylglucose alias 6²-O-α-glucosylmaltose or panose were mainlyformed from maltose as a substrate; and maltose and α-isomaltosylmaltosealias 6³-O-α-glucosylmaltotriose were mainly formed from malotriose as asubstrate along with small amounts of glucose, maltotetraose,α-isomaltosylglucose alias 6²-O-α-glucosylmaltose, and the product X.Also, it was revealed that maltotriose and the product X were mainlyformed from maltotetraose as a substrate along with small amounts ofmaltose, maltopentaose, α-isomaltosylmaltose alias6³-O-α-glucosylmaltotriose, and the product Y; and maltotetraose and theproduct Y were mainly formed from maltopentaose as a substrate alongwith small amounts of maltotriose, maltohexaose, the product X, and theproduct Z.

[0177] The product X as a main product from maltotetraose as a substrateand the product Y as a main product from maltopentaose as a substratewere respectively purified and isolated. The products X and Y wererespectively purified on HPLC using “YMC-Pack ODS-A R355-15S-15 12A”, apreparative HPLC column commercialized by YMC Co., Ltd., Tokyo, Japan,to isolate a specimen of the product X having a purity of 99.9% orhigher from the reaction product from maltotetraose in a yield of about8.3%, d.s.b., and a specimen of the product Y having a purity of 99.9%or higher from the reaction product from maltopentaose in a yield ofabout 11.5%, d.s.b.

[0178] The products X and Y were subjected to methylation analysis andNMR analysis in a usual manner. The results on their methylationanalysis are in Table 9. For the results on their NMR analyses, FIGS. 1and 2 are ¹H-NMR spectra for the products X and Y, respectively. The13C-NMR spectra for the products X and Y are FIGS. 3 and 4,respectively. The assignment of the products X and Y are tabulated inTable 10. TABLE 9 Analyzed Ratio methyl compound Product X Product Y2,3,4-trimethyl compound 1.00 1.00 2,3,6-trimethyl compound 3.05 3.982,3,4,6-tetramethyl compound 0.82 0.85

[0179] Based on these results, the product X formed from maltotetraosevia the action of the polypeptide havingα-isomaltosylglucosaccharide-forming enzyme activity was revealed as apentasaccharide, in which a glucose residue bound via α-linkage to C6-OHof glucose at the non-reducing end of maltotetraose, i.e.,α-isomaltosylmaltotriose alias 6⁴-O-α-glucosylmaltotetraose, representedby Formula 1.

α-D-Glcp-(1→6)-α-D-Glcp-(1→4)-α-D-Glcp-(1→4)-α-D-Glcp-(1→4)-α-D-Glcp  Formula 1

[0180] The product Y formed from maltopentaose was revealed as ahexasaccharide, in which a glucose residue bound via α-linkage to C6-OHof glucose at the non-reducing end of maltopentaose, i.e.,α-isomaltosylmaltotetraose alias 6⁵-O-α-glucosylmaltopentaose,represented by Formula 2.

α-D-Glcp-(1→6)-α-D-Glcp-(1→4)-α-D-Glcp-(1→4)-α-D-Glcp-(1→4)-α--D-Glcp-(1→4)-α-D-Glcp  Formula 2 TABLE 10 Glucose Carbon Chemical shift on NMR (ppm) numbernumber Product X Product Y a 1a 100.8 100.8 2a 74.2 74.2 3a 75.8 75.7 4a72.2 72.2 5a 74.5 74.5 6a 63.2 63.1 b 1b 102.6 102.6 2b 74.2 74.2 3b75.8 75.7 4b 72.1 72.1 5b 74.0 74.0 6b 68.6 68.6 c 1c 102.3 102.3 2c74.2 74.2 3c 76.0 76.0 4c 79.6 79.5 5c 73.9 73.9 6c 63.2 63.1 d 1d 102.2102.3 2d 74.0(α), 74.4(β) 74.2 3d 76.0 76.0 4d 79.8 79.5 5d 73.9 73.9 6d63.2 63.1 e 1e 94.6(α), 98.5(β) 102.1 2e 74.2(α), 76.7(β) 74.0(α),74.4(β) 3e 75.9(α), 78.9(β) 76.0 4e 79.6(α), 79.4(β) 79.8 5e 72.6(α),77.2(β) 73.9 6e 63.4(α), 63.4(β) 63.1 f 1f 94.6(α), 98.5(β) 2f 74.2(α),76.7(β) 3f 76.0(α), 78.9(β) 4f 79.6(α), 79.5(β) 5f 72.6(α), 77.2(β) 6f63.3(α), 63.3(β)

[0181] Based on these results, it was concluded that the polypeptidehaving α-isomaltosylglucosaccharide-forming enzyme activity acts onmaltooligosaccharides as shown below:

[0182] 1) The polypeptide having α-isomaltosylglucosaccharide-formingenzyme activity acts on as a substrate saccharides with a glucosepolymerization degree of two or higher and having α-1,4 glucosidiclinkage as a linkage at the non-reducing end, and catalyzes theintermolecular 6-glucosyl-transferring reaction in such a manner oftransferring a glucosyl residue at the non-reducing end of thesaccharide molecule to C-6 position of the non-reducing end of othersaccharide molecule to form both an α-isomaltosylglucosaccharide alias6-O-α-glucosylmaltooligosaccharide, having a 6-O-α-glucosyl residue anda higher glucose polymerization degree by one as compared with theintact substrate, and a maltooligosaccharide with a reduced glucosepolymerization degree by one as compared with the intact substrate; and

[0183] 2) The polypeptide having α-isomaltosylglucosaccharide-formingenzyme activity slightly catalyzes the 4-glucosyl-transferring reactionand forms small amounts of both a maltooligosaccharide, having anincreased glucose polymerization degree by one as compared with theintact substrate, and a maltooligosaccharide having a reduced glucosepolymerization degree by one as compared with the intact substrate.

[0184] Experiment 4-3

[0185] Test on Reducing-Power Formation

[0186] The following test was carried out to study whether thepolypeptide having α-isomaltosylglucosaccharide-forming enzyme activityhad the ability of forming reducing-power or not. To a 1% (w/v) aqueoussolution of maltotetraose as a substrate was added 0.25 unit/gsubstrate, d.s.b., of either of purified polypeptide specimen ofα-isomaltosylglucosaccharide-forming enzyme from Bacillus globisporusC11 obtained by the method in Experiment 1-3, Bacillus globisporus N75obtained by the method in Experiment2-3, or Arthrobacter globiformis A19obtained by the method in Experiment 3-3, and incubated at 35° C. and atpH 6.0, except that pH 8.4 was used for the enzyme from Arthrobacterglobiformis A19. During the reaction, a portion of each reaction mixturewas sampled at prescribed time intervals and measured for reducing powerafter keeping at 100° C. for 10 minutes to stop the enzymatic reaction.Before and after the enzymatic reaction, the reducing sugar content andtotal sugar content were respectively quantified by the Somogyi-Nelsonmethod and anthrone-sulfuric acid method. The percentage of formingreducing power was calculated by the following equation:

[0187] Equation:

Percentage of forming reducing power (%)=(AR/AT−BR/BT)×100

[0188] AR: Reducing sugar content after enzymatic reaction.

[0189] AT: Total sugar content after enzymatic reaction.

[0190] BR: Reducing sugar content before enzymatic reaction.

[0191] BT: Total sugar content before enzymatic reaction.

[0192] The results are in Table 11. TABLE 11 Percentage of formingreducing power (%) Reaction time Enzyme of Enzyme of Enzyme of (hour)Strain C11 Strain N75 Strain A19 0 0.0 0.0 0.0 1 0.1 0.1 0.0 2 0.0 0.00.1 4 0.1 0.0 0.0 8 0.0 0.1 0.1

[0193] As evident from the results in Table 11, it was revealed that thepolypeptide having α-isomaltosylglucosaccharide-forming enzyme activitydid not substantially increase the reducing power of the reactionproduct when acted on maltotetraose as a substrate; the polypeptidehaving α-isomaltosylglucosaccharide-forming enzyme activity did not havehydrolyzing activity or had only an undetectable level of such activity.

[0194] Experiment 4-4

[0195] Molecular Weight

[0196] Purified specimens of the polypeptide havingα-isomaltosylglucosaccharide-forming enzyme activity from Bacillusglobisporus C11 obtained by the method in Experiment 1-3, Bacillusglobisporus N75 obtained by the method in Experiment 2-3, andArthrobacter globiformis A19 obtained by the method in Experiment 3-3were subjected to SDS-PAGE using 7.5% (w/v) of polyacrylamide gel andthen determined for their molecular weights by comparing with thedynamics of standard molecular weight markers electrophoresed inparallel, commercialized by Bio-Rad Laboratories, Inc., Brussels,Belgium. It was revealed that the polypeptides from C11, N75, and A19had molecular weights of about 137,000±20,000 daltons, about136,000±20,000 daltons, and about 94,000±20,000 daltons, respectively.

[0197] Experiment 4-5

[0198] Isoelectric Point

[0199] Purified specimens of the polypeptide havingα-isomaltosylglucosaccharide-forming enzyme activity from Bacillusglobisporus C11 obtained by the method in Experiment 1-3, Bacillusglobisporus N75 obtained by the method in Experiment 2-3, andArthrobacter globiformis A19 obtained by the method in Experiment 3-3were subjected to isoelectrophoresis using a gel containing 2% (w/v)ampholine commercialized by Amersham Biosciences K. K., Tokyo, Japan(old name, Amersham Pharmacia Biotech), and then measured pHs of proteinbands and gel to determine their isoelectric points. It was revealedthat the polypeptides from C11, N75, and A19 had isoelectric points ofabout 5.2±0.5, about 7.3±0.5, and about 4.3±0.5, respectively.

[0200] Experiment 4-6

[0201] Effect of Temperature and pH

[0202] The effect of temperature and pH on theα-isomaltosylglucosaccharide-forming enzyme activity was examined inaccordance with the assay for the α-isomaltosylglucosaccharide-formingenzyme activity under various temperature and pH conditions. The effectof temperature was conducted in the presence or absence of 1 mM Ca²⁺.These results are shown in FIG. 5 (effect of temperature on thepolypeptide from a strain C11), FIG. 6 (effect of temperature on thepolypeptide from a strain N75), FIG. 7 (effect of temperature on thepolypeptide from a strain A19), FIG. 8 (effect of pH on the polypeptidefrom a strain C11), FIG. 9 (effect of pH on the polypeptide from astrain N75), and FIG. 10 (effect of pH on the polypeptide from a strainA19). The optimum temperature of the polypeptide havingα-isomaltosylglucosaccharide-forming enzyme activity from a strain C11was about 45° C. (in the absence of Ca²⁺) and about 50° C. (in thepresence of Ca²⁺) when incubated at pH 6.0 for 60 minutes, and theoptimum pH was about 6.0 when incubated at 35° C. for 60 minutes. Theoptimum temperature of the polypeptide havingα-isomaltosylglucosaccharide-forming enzyme activity from a strain N75was about 50° C. (in the absence of Ca²⁺) and about 55° C. (in thepresence of Ca²⁺) when incubated at pH 6.0 for 60 minutes, and theoptimum pH was about 6.0 when incubated at 35° C. for 60 minutes. Theoptimum temperature of the polypeptide havingα-isomaltosylglucosaccharide-forming enzyme activity from a strain A19was about 60° C. (in the absence of Ca²⁺) and about 65° C. (in thepresence of Ca²⁺) when incubated at pH 8.4 for 60 min, and the optimumpH was about 8.4 when incubated at 35° C. for 60 minutes.

[0203] Experiment 4-7

[0204] Stability

[0205] The thermal stability of the polypeptide havingα-isomaltosylglucosaccharide-forming enzyme activity was determined byincubating the testing polypeptide solution in 20 mM acetate buffer,pH6.0 (in the case of the polypeptide from a strain A19, 20 m MGlycine-NaOH buffer, pH 8.0) at prescribed temperatures for 60 minutesin the presence or absence of 1 mM Ca²⁺, cooling with water theresulting polypeptide solutions, and assaying the residual enzymeactivity of each solution. The pH stability of the polypeptide havingα-isomaltosylglucosaccharide-forming enzyme activity was determined bykeeping the testing polypeptide solutions in 50 mM buffer havingprescribed pHs at 4° C. for 24 hours, adjusting the pH of each solutionto 6.0 (in the case of the polypeptide from a strain A19, pH 8.0), andassaying the residual enzyme activity of each solution. These resultsare shown in FIG. 11 (thermal stability of the polypeptide from a strainC11), FIG. 12 (thermal stability of the polypeptide from a strain N75),FIG. 13 (thermal stability of the polypeptide from a strain A19), FIG.14 (pH stability of the polypeptide from a strain C11), FIG. 15 (pHstability of the polypeptide from a strain N75), and FIG. 16 (pHstability of the polypeptide from a strain A19). The thermal stabilityof the polypeptide having α-isomaltosylglucosaccharide-forming enzymeactivity from a strain C11 was up to about 40° C. (in the absence ofCa²⁺) and about 45° C. (in the presence of Ca²⁺), and pH stability ofabout 5.0 to about 10.0. The thermal stability of the polypeptide havingα-isomaltosylglucosaccharide-forming enzyme activity from a strain N75was up to about 45° C. (in the absence of Ca²⁺) and about 50° C. (in thepresence of Ca²⁺), and pH stability of about 5.0 to about 9.0. Thethermal stability of the polypeptide havingα-isomaltosylglucosaccharide-forming enzyme activity from a strain A19was up to about 55° C. (in the absence of Ca²⁺) and about 60° C. (in thepresence of Ca²⁺), and pH stability of about 5.0 to about 9.0.

[0206] Experiment 5

[0207] Partial Amino Acid Sequence

[0208] Experiment 5-1

[0209] N-Terminal Amino Acid Sequence

[0210] Purified specimens of the polypeptide havingα-isomaltosylglucosaccharide-forming enzyme activity from Bacillusglobisporus C11 obtained by the method in Experiment 1-3, Bacillusglobisporus N75 obtained by the method in Experiment 2-3, andArthrobacter globiformis A19 obtained by the method in Experiment 3-3were subjected to N-terminal sequence analysis by using “gas-phaseprotein sequencer model 473A”, an apparatus of Applied Biosystems, 850Lincoln Centre Drive, Foster City, U.S.A. It was revealed that thepolypeptides having α-isomaltosyl-transferring enzyme activity fromstrains C11, N75, and A19 had amino acid sequences of SEQ ID NOs:7, 19,and 26, respectively.

[0211] Experiment 5-2

[0212] Internal Amino Acid Sequences of the Polypeptide from Bacillusglobisporus C11

[0213] A part of a purified specimen of polypeptide havingα-isomaltosylglucosaccharide-forming enzyme activity, obtained inExperiment 1-3, was dialyzed against 10 mM Tris-HCl buffer (pH 9.0), andthe dialyzed solution was diluted with a fresh preparation of the samebuffer to give a concentration of about one mg/ml. One milliliter of thediluted solution as a test sample was admixed with 10 μg of trypsincommercialized by Wako Pure Chemicals, Ltd., Tokyo, Japan, and incubatedat 30° C. for 22 hours to form peptides. The resulting hydrolyzate wassubjected to revetse-phase HPLC to separate the peptides using“μ-BONDAPAK C18 column”, having a diameter of 2.1 mm and a length of 150mm, a product of Waters Chromatography Div., MILLIPORE Corp., Milford,USA, pre-equilibrated with 0.1% (v/v) trifluoroacetate containing 8%(v/v) acetonitrile, at a flow rate of 0.9 ml/minutes and at ambienttemperature, and using a linear gradient of acetonitrile increasing from8% (v/v) to 40% (v/v) in 0.1% (v/v) trifluoroacetate over 120 minutes.Peptide fragments eluted from the column were detected by monitoring theabsorbance at a wavelength of 210 nm. Ten peptide fragments, P8(Retention time (Rt): about 8 minutes), P20 (Rt: about 20 minutes), P56(Rt: about 56 minutes), P60 (Rt: about 60 minutes), P62 (Rt: about 62minutes), P64 (Rt: about 64 minutes), P75 (Rt: about 75 minutes), P82(Rt: about 82 minutes), P88 (Rt: about 88 minutes), P99 (Rt: about 99minutes), which were separated well from other peptides, were separatelycollected and dried in vacuo and then dissolved in a 200 μl solution of0.1% (v/v) trifluoroacetate and 50% (v/v) acetonitrile. Each peptidefragments had amino acid sequences of SEQ ID NOs:8 to 17 when theseamino acid sequences were analyzed by the protein sequencer used inExperiment 5-1.

[0214] Experiment 5-3

[0215] Internal Amino Acid Sequences of the Polypeptide from Bacillusglobisporus N75

[0216] A part of a purified specimen of polypeptide havingα-isomaltosylglucosaccharide-forming enzyme activity, obtained inExperiment 2-3, was dialyzed against 10 mM Tris-HCl buffer (pH 9.0), andthe dialyzed solution was diluted with a fresh preparation of the samebuffer to give a concentration of about one mg/ml. One milliliter of thediluted solution as a test sample was admixed with 20 μg of “LysylEndopeptidase” commercialized by Wako Pure Chemicals, Ltd., Tokyo,Japan, and incubated at 30° C, for 24 hours to form peptides. Theresulting hydrolyzate was subjected to reverse-phase HPLC to separatethe peptides using “μ-BONDASPHERE C18 column”, having a diameter of 3.9mm and a length of 150 mm, a product of Waters Chromatography Div.,MILLIPORE Corp., Milford, USA, pre-equilibrated with 0.1% (v/v)trifluoroacetate containing 8% (v/v) acetonitrile, at a flow rate of 0.9ml/minutes and at ambient temperature, and using a linear gradient ofacetonitrile increasing from 8% (v/v) to 36% (v/v) in 0.1% (v/v)trifluoroacetate over 120 minutes. Peptide fragments eluted from thecolumn were detected by monitoring the absorbance at a wavelength of 210nm. Five peptide fragments, PN47 (Rt: about 47 minutes), PN59 (Rt: about59 minutes), PN67 (Rt: about 67 minutes), PN87 (Rt: about 87 minutes),PN89 (Rt: about 89 minutes), which were separated well from otherpeptides, were separately collected and dried In vacuo and thendissolved in a 200 μl solution of 0.1% (v/v) trifluoroacetate and 50%(v/v) acetonitrile. Each peptide fragments had amino acid sequences ofSEQ ID NOs:20 to 24 when these amino acid sequences were analyzed by theprotein sequencer used in Experiment 5-1.

[0217] Experiment 5-4

[0218] Internal Amino Acid Sequences of the Polypeptide fromArthrobacter globiformis A19

[0219] A part of a purified specimen of polypeptide havingα-isomaltosylglucosaccharide-forming enzyme activity, obtained inExperiment 3-3, was dialyzed against 10 mM Tris-HCl buffer (pH 9.0), andthe dialyzed solution was diluted with a fresh preparation of the samebuffer to give a concentration of about one mg/ml. One milliliter of thediluted solution as a test sample was admixed with 20 μg of “LysylEndopeptidase” commercialized by Wako Pure Chemicals, Ltd., Tokyo,Japan, and incubated at 30° C. for 24 hours to form peptides. Theresulting hydrolyzate was subjected to reverse-phase HPLC to separatethe peptides using “μ-BONDASPHERE C18 column”, having a diameter of 2.1mm and a length of 150 mm, a product of Waters Chromatography Div.,MILLIPORE Corp., Milford, USA, pre-equilibrated with 0.1% (v/v)trifluoroacetate containing 16% (v/v) acetonitrile, at a flow rate of0.9 ml/minutes and at ambient temperature, and using a linear gradientof acetonitrile increasing from 16% (v/v) to 36% (v/v) in 0.1% (v/v)trifluoroacetate over 120 minutes. Peptide fragments eluted from thecolumn were detected by monitoring the absorbance at a wavelength of 210nm. Five peptide fragments, PA39 (Rt: about 39 minutes), PA81 (Rt: about81 minutes), PA86 (Rt: about 86 minutes), PA92 (Rt: about 92 minutes),PA104 (Rt: about 104 minutes), which were separated well from otherpeptides, were separately collected and dried in vacuo and thendissolved in a 200 μl solution of 0.1% (v/v) trifluoroacetate and 50%(v/v) acetonitrile. Each peptide fragments had amino acid sequences ofSEQ ID NOs:27 to 31 when these amino acid sequences were analyzed by theprotein sequencer used in Experiment 5-1.

[0220] Experiment 6

[0221] Preparation of a Recombinant DNA Containing a DNA EncodingPolypeptide from Bacillus globisporus C11 and a Transformant

[0222] Experiment 6-1

[0223] Preparation of Chromosomal DNA

[0224] A liquid culture medium consisting 2% (w/v) of “PINE-DEX #4”, apartial starch hydrolyzate, 1.0% (w/v) of “ASAHIMEAST”, a yeast extract,0.1% (w/v) of dipotassium phosphate, 0.06% (w/v) of sodium phosphatedodeca-hydrate, 0.05% (w/v) of magnesium sulfate hepta-hydrate, andwater was placed in 500-ml Erlenmeyer flasks in a respective amount of100 ml, sterilized by autoclaving at 121° C. for 20 minutes, cooled andinoculated with Bacillus globisporus C11, followed by culturing underrotary-shaking conditions at 27° C. and 230 rpm for 24 hours. The cellscollected from the culture by centrifugation were suspended in TESbuffer (pH 8.0), the suspended solution was admixed with lysozyme togive a concentration of 0.05% (w/v), and incubated at 37° C. for 30minutes. After freezing the lysate at −80° C. for one hour, the lysatewas added with TSS buffer (pH 9.0)and heated to 60° C. The solution wasadded with a mixture of TES buffer and phenol, and was vigorously shookfor five minute in an ice bath, and the supernatant was collected bycentrifugation. The supernatant was added twice volume of cold ethanol,and resulting crude precipitate was collected as a crude chromosomalDNA. The crude chromosomal DNA was dissolved in SSC buffer (pH 7.1), andadmixed with 7.5 μg of ribonuclease and 125 μg of proteinase, andincubated 37° C. for one hour. The chromosomal DNA was extracted fromthe reactant by adding chloroform/isoamylalcohol mixture, then addedcold ethanol, and the resulting precipitate containing chromosomal DNAwas collected. The purified chromosomal DNA, obtained according to themethod described above, was dissolved in SSC buffer (pH 7.1) to give aconcentration of about one mg/ml and frozen at −80° C.

[0225] Experiment 6-2

[0226] Preparation of a Transformant. BGC2

[0227] One milliliter of purified chromosomal DNA solution, prepared bythe method in Experiment 6-1, was admixed with about 35 units of arestriction enzyme, Sau 3AI, and incubated at 37° C. for 20 minutes forpartial digestion of the chromosomal DNA. The resulting DNA fragmentscorresponding to about 2,000 to 6,000 base pairs were collected bysucrose density-gradient centrifugation. A plasmid vector, Bluescript IISK(+), commercialized by Stratagene Cloning System, was completelydigested with a restriction enzyme, Bam HI by conventional method. Arecombinant DNA was obtained by ligating 0.5 μg of the digested plasmidvector with about 5 μg of the DNA fragments prepared before by using a“DNA ligation kit”, commercialized by Takara Shuzo Co., Ltd., accordingto the method described in a document attached with the kit. Then, agene library was prepared by transforming 100 μl portion of thecompetent cell, “Epicurian Coli XL2-Blue”, commercialized by StratageneCloning System, with the recombinant DNA by conventional competent cellmethod. The transformants thus obtained as gene library were inoculatedinto a fresh agar plate medium (pH 7.0) containing 10 g/L of tryptone, 5g/L of yeast extract, 5 g/L of sodium chloride, 100 mg/L of ampicillinsodium salt, and 50 mg/L of 5-bromo-4-chloro-3-indolyl-β-galactoside,and incubated at 37° C. for 24 hours. About five thousand white coloniesgrown on the plate were transferred to and fixed on a nylon membrane,“Hybond-N+”, commercialized by Amasham Bioscience K. K. Anoligonucleotide having a nucleotide sequence of“5′-GGNTTYATGAAYTTYAGRTGGGA-3′” was chemically synthesized on the basesof an amino acid sequence of fourth to eleventh of SEQ ID NO:16, whichdisclosed by the method in Experiment 5-2. A synthetic DNA (probe 1) wasobtained by labeling the oligonucleotide with radioisotope using[γ-³²P]ATP and T4 polynucleotide kinase according to the conventionalmethod. Subsequently, two types of transformants showing remarkablehybridization with probe 1 were selected from the colonies fixed on thenylon membrane obtained before, using conventional colony hybridization.The recombinant DNAs were collected from these two types oftransformants by conventional method. On the other hand, probe 2 havingthe nucleotide sequence of “5′-GAYGCNTGGATGTTYGGNGAYTGG-3′” waschemically synthesized based on a amino acid sequence of fourth toeleventh of SEQ ID NO:17 and labeled with radioisotope in the samemanner. The recombinant DNAs obtained and probe 2 were used forconventional southern-hybridization, and a recombinant DNA showing aremarkable hybridization with probe 2 was selected. A transformant thusselected was named “BGC2”.

[0228] Experiment 6-3

[0229] Analysis of DNA Sequence

[0230] According to the conventional method, a transformant, BGC2,obtained by the method in Experiment 6-2, was inoculated into L-brothmedium (pH 7.0) containing 100 μg/ml of ampicillin sodium salt, andcultured under rotary-shaking conditions at 37° C. for 24 hours. Afterthe completion of the culture, cells were collected by centrifugation,and the recombinant DNA was extracted from the cells by conventionalalkaline-SDS method. When the nucleotide sequence of the recombinant DNAwas analyzed by conventional dideoxy method, it was revealed that therecombinant DNA contained a DNA having the nucleotide sequence of SEQ IDNO:18, 5,294 base pairs, which originated from Bacillus globisporus C11.In the recombinant DNA, as shown in FIG. 17, the DNA was ligated atdownstream of recognition site of a restriction enzyme, Xba I. The aminoacid sequence deduced from the nucleotide sequence is as shown inparallel in SEQ ID NO:18. The amino acid sequence was compared withamino acid sequences of polypeptide of the present invention, i.e., theN-terminal amino acid sequence of SEQ ID NO:7 disclosed by the method inExperiment 5-1 and the internal partial amino acid sequences of SEQ IDNO:8 to 17 disclose by the method in Experiment 5-2. An amino acidsequence of SEQ ID NO:7 was completely identical with that of 36th to44th of the amino acid sequence shown in parallel in SEQ ID NO:18. Aminoacid sequences of SEQ ID NOs:8, 9, 10, 11, 12,13, 14, 15, 16, and 17were completely identical with those of 823rd to 832nd, 576th to 589th,874th to 904th, 1117th to 1141st, 657th to 670th, 367th to 399th, 970thto 993rd, 938th to 953rd, 279th to 295th, and 632nd to 651st of theamino acid sequence shown in parallel in SEQ ID NO:18, respectively.Since the nucleotide sequence of 4,783rd to 4785th of SEQ ID NO:18encodes a codon for terminating the translation (stop codon, 5′-TAA-3′),it is revealed that C-terminal amino acid of the polypeptide of thepresent invention is glutamic acid (1,284th amino acid of the amino acidsequence shown in parallel in SEQ ID NO:18). These results indicate thatthe polypeptide of the present invention contains the amino acidsequence of SEQ ID NO:1, and that the polypeptide is encoded by the DNAhaving the nucleotide sequence of SEQ ID NO:4 in the case of Bacillusglobisporus C11. An amino acid sequence of the first to 35th of thatshowing in parallel in SEQ ID NO:18 was presumed to be a secretionsignal sequence of the polypeptide. According to the results describedabove, it was revealed that the precursor peptide of the polypeptidebefore secretion had the amino acid sequence shown in parallel in SEQ IDNO:18, and the amino acid sequence was encoded by the nucleotidesequence of SEQ ID NO:18. The recombinant DNA prepared and confirmed thenucleotide sequence as described above was named “pBGC2”.

[0231] Experiment 7

[0232] Preparation of a Recombinant DNA Containing a DNA EncodingPolypeptide from Bacillus globisporus N75 and a Transformant

[0233] Experiment 7-1

[0234] Preparation of Chromosomal DNA

[0235] A liquid culture medium consisting 2% (w/v) of “PINE-DEX #4”, apartial starch hydrolyzate, 1.0% (w/v) of “ASAHIMEAST”, a yeast extract,0.1% (w/v) of dipotassium phosphate, 0.06% (w/v) of sodium phosphatedodeca-hydrate, 0.05% (w/v) of magnesium sulfate hepta-hydrate, andwater was placed in 500-ml Erlenmeyer flasks in a respective amount of100 ml, sterilized by autoclaving at 121° C. for 20 minutes, cooled andinoculated with Bacillus globisporus N75, followed by culturing underrotary-shaking conditions at 27° C. and 230 rpm for 24 hours. The cellscollected from the culture by centrifugation were suspended in TESbuffer (pH 8.0), the suspended solution was admixed with lysozyme togive a concentration of 0.05% (w/v), and incubated at 37° C. for30minutes. After freezing the lysate at −80° C. for one hour, the lysatewas added with TSS buffer (pH 9.0)and heated to 60° C. The solution wasadded with a mixture of TES buffer and phenol, and was vigorously shookfor five minute in an ice bath, and the supernatant was collected bycentrifugation. The supernatant was added twice volume of cold ethanol,and resulting crude precipitate was collected as crude chromosomal DNA.The crude chromosomal DNA was dissolved in SSC buffer (pH 7.1), andadmixed with 7.5 μg of ribonuclease and 125 μg of proteinase, andincubated 37° C. for one hour. The chromosomal DNA was extracted fromthe reactant by adding chloroform/isoamylalcohol mixture, then addedcold ethanol, and the resulting precipitate containing chromosomal DNAwas collected. The purified chromosomal DNA, obtained according to themethod described above, was dissolved in SSC buffer (pH 7.1) to give aconcentration of about one mg/ml and frozen at −80° C.

[0236] Experiment 7-2

[0237] Preparation of a Transformant, BGN2

[0238] One hundred μl (0.1 ml) of purified chromosomal DNA solution,prepared by the method in Experiment 7-1, was admixed with about 200units of a restriction enzyme, Kpn I, and incubated at 37° C. for 16hours to digest the chromosomal DNA. The resulting DNA fragments wereseparated by agarose gel electrophoresis, and DNA fragmentscorresponding to about 3,000 to 7,000 base pairs were collected. Aplasmid vector, Bluescript II SK(+), commercialized by StratageneCloning System, was completely digested with a restriction enzyme, KpnI. A recombinant DNA was obtained by ligating 0.5 μg of the digestedplasmid vector with about 5 μg of the DNA fragments prepared before byusing a “DNA ligation kit”, commercialized by Takara Shuzo Co., Ltd.,according to the method described in a document attached with the kit.Then, a gene library was prepared by transforming 100 μl portion of thecompetent cell, “Epicurian Coli XL2-Blue”, commercialized by StratageneCloning System, with the recombinant DNA by conventional competent cellmethod. The transformants thus obtained as gene library were inoculatedinto a fresh agar plate medium (pH 7.0) containing 10 g/L of tryptone, 5g/L of yeast extract, 5 g/L of sodium chloride, 100 mg/L of ampicillinsodium salt, and 50 mg/L of 5-bromo-4-chloro-3-indolyl-β-galactoside,and incubated at 37° C. for 24 hours. About 2,500 white colonies grownon the plate were transferred to and fixed on a nylon membrane,“Hybond-N+”, commercialized by Amasham Bioscience K. K. Anoligonucleotide having a nucleotide sequence of“5′-GAYGCNTGGATGTTYGGNGAYTGG-3′” was chemically synthesized on the basesof an amino acid sequence of fourth to eleventh of SEQ ID NO:24, whichdisclosed by the method in Experiment 5-3. A synthetic DNA (probe 1) wasobtained by labeling the oligonucleotide with radioisotope using[γ-³²P]ATP and T4 polynucleotide kinase according to the conventionalmethod. Subsequently, three types of transformant showing remarkablehybridization with probe 1 were selected from the colonies fixed on thenylon membrane obtained before, using conventional colony hybridization.The recombinant DNAs were collected from these three types oftransformant by conventional method. On the other hand, probe 2 havingthe nucleotide sequence of “5′-GTNAAYCARAAYCAYTGGTTYTA-3′” waschemically synthesized based on a amino acid sequence of fourteenth totwenty-first of SEQ ID NO:23 and labeled with radioisotope in the samemanner. The recombinant DNAs obtained and probe 2 were used forconventional southern-hybridization, and a recombinant DNA showing aremarkable hybridization with probe 2 was selected. A transformant thusselected was named “BGN2”.

[0239] Experiment 7-3

[0240] Analysis of DNA Sequence

[0241] According to the conventional method, the transformant, BGN2,obtained by the method in Experiment 7-2, was inoculated into L-brothmedium (pH 7.0) containing 100 μg/ml of ampicillin sodium salt, andcultured under rotary-shaking conditions at 37° C. for 24 hours. Afterthe completion of the culture, cells were collected by centrifugation,and the recombinant DNA was extracted from the cells by conventionalalkaline-SDS method. When the nucleotide sequence of the recombinant DNAwas analyzed by conventional dideoxy method, it was revealed that therecombinant DNA contained a DNA having the nucleotide sequence of SEQ IDNO:25, 4,991 base pairs, which originated from Bacillus globisporus N75.In the recombinant DNA, as shown in FIG. 18, the DNA was ligated atdownstream of recognition site of a restriction enzyme, Kpn I. The aminoacid sequence deduced from the nucleotide sequence is as shown inparallel in SEQ ID NO:25. The amino acid sequence was compared withamino acid sequences of polypeptide of the present invention, i.e., theN-terminal amino acid sequence of SEQ ID NO:19 disclosed by the methodin Experiment 5-1 and the internal partial amino acid sequences of SEQID NOs:20 to 24 disclosed by the method in Experiment 5-3. An amino acidsequence of SEQ ID NO:19 was completely identical with that of 36th to43rd of the amino acid sequence shown in parallel in SEQ ID NO:25. Aminoacid sequences of SEQ ID NOs:20, 21, 22, 23, and24were completelyidentical with those of 907th to925th, 367th to 386th, 1,034th to1,058th, 996th to 1,020th, and 632nd to 642nd of the amino acid sequenceshown in parallel in SEQ ID NO:25, respectively. Since the nucleotidesequence of 4,294th to 4,296th of SEQ ID NO:25encodes a codon forterminating the translation (stop codon, 5′-TAA-3′), it is revealed thatC-terminal amino acid of the polypeptide of the present invention isglutamine (1,286th amino acid of the amino acid sequence shown inparallel in SEQ ID NO: 25). These results indicate that the polypeptideof the present invention contains the amino acid sequence of SEQ IDNO:2, and that the polypeptide is encoded by the DNA having thenucleotide sequence of SEQ ID NO: 5 in the case of Bacillus globisporusN75. An amino acid sequence of the first to 35th of that showing inparallel in SEQ ID NO: 25 was presumed to be a secretion signal sequenceof the polypeptide. According to the results described above, it wasrevealed that the precursor peptide of the polypeptide before secretionhad the amino acid sequence shown in parallel in SEQ ID NO:25, and theamino acid sequence was encoded by the nucleotide sequence of SEQ IDNO:25. The recombinant DNA prepared and confirmed the nucleotidesequence as described above was named “pBGN2”.

[0242] Experiment 8

[0243] Preparation of a Recombinant DNA Containing a DNA EncodingPolypeptide from Arthrobacter globiformis A19 and a Transformant

[0244] Experiment 8-1

[0245] Preparation of Chromosomal DNA

[0246] A liquid culture medium consisting 2% (w/v) of “PINE-DEX #4”, apartial starch hydrolyzate, 1.0% (w/v) of “ASAHIMEAST”, a yeast extract,0.1% (w/v) of dipotassium phosphate, 0.06% (w/v) of sodium phosphatedodeca-hydrate, 0.05% (w/v) of magnesium sulfate hepta-hydrate, andwater was placed in 500-ml Erlenmeyer flasks in a respective amount of100 ml, sterilized by autoclaving at 121° C. for 20 minutes, cooled andinoculated with Arthrobacter globiformis A19, followed by culturingunder rotary-shaking conditions at 27° C. and 230 rpm for 24 hours. Thecells collected from the culture by centrifugation were suspended in TESbuffer (pH 8.0), the suspended solution was admixed with lysozyme togive a concentration of 0.05% (w/v), and incubated at 37° C. for 30minutes. After freezing the lysate at −80° C. for one hour, the lysatewas added with TSS buffer (pH 9.0)and heated to 60° C. The solution wasadded with a mixture of TES buffer and phenol, and was vigorously shookfor five minute in an ice bath, and the supernatant was collected bycentrifugation. The supernatant was added twice volume of cold ethanol,and resulting crude precipitate was collected as crude chromosomal DNA.The crude chromosomal DNA was dissolved in SSC buffer (pH 7.1), andadmixed with 7.5 μg of ribonuclease and 125 μg of proteinase, andincubated 37° C. for one hour. The chromosomal DNA was extracted fromthe reactant by adding chloroform/isoamylalcohol mixture, then addedcold ethanol, and the resulting precipitate containing chromosomal DNAwas collected. The purified chromosomal DNA, obtained according to themethod described above, was dissolved in SSC buffer (pH 7.1) to give aconcentration of about one mg/ml and frozen at −80° C.

[0247] Experiment 8-2

[0248] Preparation of a Transformant. AGA1

[0249] One ml of purified chromosomal DNA solution, prepared by themethod in Experiment 8-1, was admixed with about 10 units of arestriction enzyme, Kpn I, and incubated at 37° C. for 30 minutes todigest partially the chromosomal DNA. The resulting DNA fragments wereseparated by agarose gel electrophoresis, and DNA fragmentscorresponding to about 4,000 to 8,000 base pairs were collected. Aplasmid vector, Bluescript II SK(+), commercialized by StratageneCloning System, was completely digested with a restriction enzyme, KpnI. A recombinant DNA was obtained by ligating 0.5 μg of the digestedplasmid vector with about 5 μg of the DNA fragments prepared before byusing a “DNA ligation kit”, commercialized by Takara Shuzo Co., Ltd.,according to the method described in a document attached with the kit.Then, a gene library was prepared by transforming 100 μl portion of thecompetent cell, “Epicurian Coli XL2-Blue”, commercialized by StratageneCloning System, with the recombinant DNA by conventional competent cellmethod. The transformants thus obtained as gene library were inoculatedinto a fresh agar plate medium (pH 7.0) containing 10 g/L of tryptone, 5g/L of yeast extract, 5 g/L of sodium chloride, 100 mg/L of ampicillinsodium salt, and 50 mg/L of 5-bromo-4-chloro-3-indolyl-β-galactoside,and incubated at 37° C. for 24 hours. About six thousands white coloniesgrown on the plate were transferred to and fixed on a nylon membrane,“Hybond-N+”, commercialized by Amasham Bioscience K. K. Anoligonucleotide having a nucleotide sequence of“5′-CARGARTGGAAYYTNACNGGNGAYCCNTGGAC-3′” was chemically synthesized onthe bases of an amino acid sequence of first to eleventh of SEQ IDNO:27, which disclosed by the method in Experiment 5-4. A synthetic DNA(probe 1) was obtained by labeling the oligonucleotide with radioisotopeusing [γ-³²P]ATP and T4 polynucleotide kinase according to theconventional method. Subsequently, two types of transformant showingremarkable hybridization with probe 1 were selected from the coloniesfixed on the nylon membrane obtained before, using conventional colonyhybridization. The recombinant DNAs were collected from these two typesof transformant by conventional method. On the other hand, probe 2having the nucleotide sequence of“5′-TGGACNCARCCNGARGCNGGNGCNGTNTTGCA-3′” was chemically synthesizedbased on a amino acid sequence of sixth to sixteenth of SEQ ID NO: 29and labeled with radioisotope in the same manner. The recombinant DNAsobtained and probe 2 were used for conventional southern-hybridization,and a recombinant DNA showing a remarkable hybridization with probe 2was selected. A transformant thus selected was named “AGA1”.

[0250] Experiment 8-3

[0251] Analysis of DNA Sequence

[0252] According to the conventional method, the transformant, AGA1,obtained by the method in Experiment 8-2, was inoculated into L-brothmedium (pH 7.0) containing 100 μg/ml of ampicillin sodium salt, andcultured under rotary-shaking conditions at 37° C. for 24 hours. Afterthe completion of the culture, cells were collected by centrifugation,and the recombinant DNA was extracted from the cells by conventionalalkaline-SDS method. When the nucleotide sequence of the recombinant DNAwas analyzed by conventional dideoxy method, it was revealed that therecombinant DNA contained a DNA having the nucleotide sequence of SEQ IDNO:32, 5,811 base pairs, which originated from Arthrobacter globiformisA19. In the recombinant DNA, as shown in FIG. 19, the DNA was ligated atdownstream of recognition site of a restriction enzyme, Kpn I. The aminoacid sequence deduced from the nucleotide sequence is as shown inparallel in SEQ ID NO:32. The amino acid sequence was compared withamino acid sequences of polypeptide of the present invention, i.e., theN-terminal amino acid sequence of SEQ ID NO:26 disclosed by the methodin Experiment 5-1 and the internal partial amino acid sequences of SEQID NOs:27 to 31 disclosed by the method in Experiment 5-4. An amino acidsequence of SEQ ID NO:26 was completely identical with that of 37th to49th of the amino acid sequence shown in parallel in SEQ ID NO:32. Aminoacid sequences of SEQ ID NOs:27, 28, 29, 30, and 31 were completelyidentical with those of 227th to 239th, 345th to 374th, 401st to 430th,89th to 115th, and 641st to 667th of the amino acid sequence shown inparallel in SEQ ID NO: 32, respectively. Since the nucleotide sequenceof 4,550th to 4,552nd of SEQ ID NO:32 encodes a codon for terminatingthe translation (stop codon, 5′-TGA-3′), it is revealed that C-terminalamino acid of the polypeptide of the present invention is phenylalanine(965th amino acid of the amino acid sequence shown in parallel in SEQ IDNO:32).

[0253] These results indicate that the polypeptide of the presentinvention contains the amino acid sequence of SEQ ID NO:3, and that thepolypeptide is encoded by the DNA having the nucleotide sequence of SEQID NO:6 in the case of Arthrobacter globiformis A19 (FERM BP-7590). Anamino acid sequence of the first to 36th of that showing in parallel inSEQ ID NO:32 was presumed to be a secretion signal sequence of thepolypeptide. According to the results described above, it was revealedthat the precursor peptide of the polypeptide before secretion had theamino acid sequence shown in parallel in SEQ ID NO:32, and the aminoacid sequence was encoded by the nucleotide sequence of SEQ ID NO:32.The recombinant DNA prepared and confirmed the nucleotide sequence asdescribed above was named “pAGA1”.

[0254] Experiment 9

[0255] Production of Polypeptides by Transformants of the PresentInvention

[0256] Experiment 9-1

[0257] A Transformant. BGC2

[0258] A liquid culture medium consisting 5 g/L of “PINE-DEX #4”, apartial starch hydrolyzate, 20 g/L of polypeptone, 20 g/L of yeastextract, 1 g/L of sodium phosphate dodeca-hydrate, and water was placedin a 500-ml Erlenmeyer flask in a amount of 100 ml, sterilized byautoclaving at 121° C. for 15 minutes, and cooled. Then, the liquidmedium was sterilely set to pH 7.0, and sterilely admixed with 10 mg ofampicillin sodium salt. A transformant, BGC2, obtained by the method inExperiment 6-2, was inoculated into the above liquid medium, andcultured at 27° C. and for 48 hours under aeration-agitation conditions.To investigate the location of the polypeptide in the culture, cells andsupernatant were separately collected by conventional centrifugation. Inthe case of the cells, whole-cell extract, obtained by ultrasonicdisruption, and periplasmic extract, obtained by osmotic shock procedurewere prepared separately. In the case of ultrasonic disruption, cellswere suspended in 10 mM sodium phosphate buffer (pH 7.0), and thendisrupted in a ice bath using a ultrasonic homogenizer, “model UH-600”,commercialized by MST Corporation, Aichi, Japan, to extract whole-cellfraction. In the case of osmotic shock procedure, cells were washed with10 mM Tris-HCl buffer (pH 7.3) containing 30 mM sodium chloride, and thewashed cells were suspended in 33 mM Tris-HCl buffer (pH 7.3) containing200 g/L of sucrose and 1 mM EDTA, shook at 27° C. for 20 minutes, andthen centrifuged to collect the cells. Subsequently, the cells weresuspended in 0.5 mM magnesium chloride solution pre-cooled at about 4°C., and shook in ice bath for 20 minutes to extract periplasmicfraction. After centrifuging, the resulting supernatant was collected asperiplasmic extract.

[0259] α-Isomaltosylglucosaccharide-forming enzyme activities of culturesupernatant, whole-cell extract and periplasmic extract, prepared asdescribed above, were assayed, and those values were expressed in termsof the activities/ml-culture, respectively. The results are shown inTable 12. TABLE 12 α-isomaltosylglucosaccharide- forming enzyme activitySample (units/ml-culture) Culture supernatant 0.0 Whole-cell extract 1.1Periplasmic extract 1.0

[0260] As evident from the results in Table 12, it was revealed that thetransformant, BGC2, produced the polypeptide of the present inventionintracellularly, and secreted most of it in periplasmic fraction. As thefirst control experiment, E. coli XL2-Blue was cultured with the sameconditions in the case of the transformant described above except forthe addition of ampicillin, and a supernatant and a cell-extract wereprepared from the culture. As the second control experiment, Bacillusglobisporus C11, was cultured with the same conditions in the case ofthe transformant described above except for the addition of ampicillin,and a supernatant and a cell-extract were prepared from the culture. Inthe first control experiment, the enzyme activity was not detected fromeither of the culture supernatant and the cell-extract. In the secondcontrol experiment, the enzyme activity of the culture supernatant andthe cell-extract were about 0.37 units and about 0.02 units,respectively. Compared with the case of the transformant BGC2, theenzyme activity was evidently low-level values.

[0261] The periplasmic fraction was further purified by salting out,dialysis and successive column chromatographies on “SEPABEADS FP-DA13”gel, “SEPHACRYL HR S-200” gel, and “BUTYL-TOYOPEARL 650M” gel accordingto the methods described in Experiment 1, and the purified polypeptidewas analyzed according to the methods described in Experiment 1. As theresults, the molecular weight was about 137,000±2,000 daltons bySDS-polyacrylamide gel electrophoresis, the isoelectric point was about5.2±0.5 by polyacrylamide gel isoelectrophoresis, the optimumtemperature of α-isomaltosyl-transferring enzyme activity was about 45°C. (in the absence of Ca²⁺) and about 50° C. (in the presence of Ca²⁺),the optimum pH was about 6.0, the thermal stability was up to about 40°C. (in the absence of Ca²⁺) and about 50° C. (in the presence of Ca²⁺),and the pH stability was in the range of about pH 5.0 to about 10.0.These physicochemical properties were practically identical to those ofthe polypeptide having α-isomaltosylglucosaccharide-forming enzymeactivity prepared in Experiment 1.

[0262] Experiment 9-2

[0263] A Transformant. BGN2

[0264] A liquid culture medium consisting 5 g/L of “PINE-DEX #4”, apartial starch hydrolyzate, 20 g/L of polypeptone, 20 g/L of yeastextract, 1 g/L of sodium phosphate dodeca-hydrate, and water was placedin a 500-ml Erlenmeyer flask in a amount of 100 ml, sterilized byautoclaving at 121° C. for 15 minutes, and cooled. Then, the liquidmedium was sterilely set to pH 7.0, and sterilely admixed with 10 mg ofampicillin sodium salt. A transformant, BGN2, obtained by the method inExperiment 7-2, was inoculated into the above liquid medium, andcultured at 27° C. and for 48 hours under aeration-agitation conditions.To investigate the location of the polypeptide in the culture, culturesupernatant, whole-cell extract, and periplasmic extract were separatelyprepared according to the method in Experiment 9-1.α-Isomaltosylglucosaccharide-forming enzyme activities of culturesupernatant, whole-cell extract and periplasmic extract were assayed,and those values were expressed in terms of the activities/ml-culture,respectively. The results are shown in Table 13. TABLE 13α-isomaltosylglucosaccharide- forming enzyme activity Sample(units/ml-culture) Culture supernatant 0.54 Whole-cell extract 0.91Periplasmic extract 0.85

[0265] As evident from the results in Table 13, it was revealed that thetransformant, BGN2, produced the polypeptide of the present inventionintracellularly, and secreted most of it in periplasmic fraction. As thefirst control experiment, E. coli XL2-Blue was cultured with the sameconditions in the case of the transformant described above except forthe addition of ampicillin, and a supernatant and a cell-extract wereprepared from the culture. As the second control experiment, Bacillusglobisporus N75, was cultured with the same conditions in the case ofthe transformant described above except for the addition of ampicillin,and a supernatant and a cell-extract were prepared from the culture. Inthe first control experiment, the enzyme activity was not detected fromeither of the culture supernatant and the cell-extract. In the secondcontrol experiment, the enzyme activity of the culture supernatant andthe cell-extract were about 0.21 units and about 0.01 units,respectively. Compared with the case of the transformant BGN2, theenzyme activity was evidently low-level values.

[0266] The periplasmic fraction was further purified by salting out,dialysis and successive column chromatographies on “SEPABEADS FP-DA13”gel, “SEPHACRYL HR S-200” gel, and “BUTYL-TOYOPEARL 650M” gel accordingto the methods described in Experiment 2, and the purified polypeptidewas analyzed according to the methods described in Experiment 2. As theresults, the molecular weight was about 136,000±2,000 daltons bySDS-polyacrylamide gel electrophoresis, the isoelectric point was about7.3±0.5 by polyacrylamide gel isoelectrophoresis, the optimumtemperature of α-isomaltosyl-transferring enzyme activity was about 50°C. (in the absence of Ca²⁺) and about 55° C. (in the presence of Ca²⁺),the optimum pH was about 6.0, the thermal stability was up to about 45°C. (in the absence of Ca²⁺) and about 50° C. (in the presence of Ca²⁺),and the pH stability was in the range of about pH 5.0 to about 9.0.These physicochemical properties were practically identical to those ofthe polypeptide having α-isomaltosylglucosaccharide-forming enzymeactivity prepared in Experiment 2.

[0267] Experiment 9-3

[0268] A Transformant. AGA1

[0269] A liquid culture medium consisting 5 g/L of “PINE-DEX #4”, apartial starch hydrolyzate, 20. g/L of polypeptone, 20 g/L of yeastextract, 1 g/L of sodium phosphate dodeca-hydrate, and water was placedin a 500-ml Erlenmeyer flask in a amount of 100 ml, sterilized byautoclaving at 121° C. for 15 minutes, and cooled. Then, the liquidmedium was sterilely set to pH 7.0, and sterilely admixed with 10 mg ofampicillin sodium salt. A transformant, AGA1, obtained by the method inExperiment 8-2, was inoculated into the above liquid medium, andcultured at 27° C. and for 48 hours under aeration-agitation conditions.To investigate the location of the polypeptide in the culture, culturesupernatant, whole-cell extract, and periplasmic extract were separatelyprepared according to the method in Experiment 9-1.α-Isomaltosylglucosaccharide-forming enzyme activities of culturesupernatant, whole-cell extract and periplasmic extract were assayed,and those values were expressed in terms of the activities/ml-culture,respectively. The results are shown in Table 14. TABLE 14α-isomaltosylglucosaccharide- forming enzyme activity Sample(units/ml-culture) Culture supernatant 0.51 Whole-cell extract 2.5Periplasmic extract 2.4

[0270] As evident from the results in Table 14, it was revealed that thetransformant, AGA1, produced the polypeptide of the present inventionintracellularly, and secreted most of it in periplasmic fraction. As thefirst control experiment, E. coli XL2-Blue was cultured with the sameconditions in the case of the transformant described above except forthe addition of ampicillin, and a supernatant and a cell-extract wereprepared from the culture. As the second control experiment,Arthrobacter globiformis A19, was cultured with the same conditions inthe case of the transformant described above except for the addition ofampicillin, and a supernatant and a cell-extract were prepared from theculture. In the first control experiment, the enzyme activity was notdetected from either of the culture supernatant and the cell-extract. Inthe second control experiment, the enzyme activity of the culturesupernatant and the cell-extract were about 0.33 units and about 0.01units, respectively. Compared with the case of the transformant AGA1,the enzyme activity was evidently low-level values.

[0271] The periplasmic fraction was further purified by salting out,dialysis and successive column chromatographies on “DEAE-TOYOPEARL 650M”gel and “SEPHACRYL HR S-200” gel according to the methods described inExperiment 3, and the purified polypeptide was analyzed according to themethods described in Experiment 3. As the results, the molecular weightwas about 94,000±2,000 daltons by SDS-polyacrylamide gelelectrophoresis, the isoelectric point was about 4.3±0.5 bypolyacrylamide gel isoelectrophoresis, the optimum temperature ofα-isomaltosyl-transferring enzyme activity was about 60° C. (in theabsence of Ca²⁺) and about 65° C. (in the presence of Ca²⁺), the optimumpH was about 8.4, the thermal stability was up to about 55° C. (in theabsence of Ca²⁺) and about 60° C. (in the presence of Ca²⁺), and the pHstability was in the range of about pH 5.0 to about 9.0. Thesephysicochemical properties were practically identical to those of thepolypeptide having α-isomaltosylglucosaccharide-forming enzyme activityprepared in Experiment 3.

[0272] From the results described above, it is revealed that thepolypeptide of the present invention can be produced by recombinant DNAtechniques, and that the productivity of the polypeptide is remarkablyincreased.

[0273] The following examples concretely explain the productionprocesses for the polypeptide of the present invention,cyclotetrasaccharide obtainable thereby, and saccharides comprising thesame:

EXAMPLE 1

[0274] Production of the Polypeptide of the Present Invention

[0275] A liquid medium containing 5 g/L of “PINE-DEX #4”, a partialstarch hydrolyzate, 20 g/L of polypeptone, 20 g/L of yeast extract, 1g/L of sodium phosphate, and water was placed in a 500-ml Erlenmeyerflask in an amount of 100 ml, sterilized at 121° C. for 15 minutes, andcooled. Then, the liquid medium was sterilely set to pH 7.0, and admixedwith ampicillin sodium salt to give a final concentration of 100 μg/ml.A transformant, BGC2, obtained by the method in Experiment 5-2, wasinoculated into the above liquid medium, and cultured at 27° C. and at230 rpm for 24 hours to obtain the seed culture. Subsequently, about 18L of a fresh preparation of the same liquid culture medium as used aboveseed culture was placed in a 30-L fermentor, sterilized with the samemanner, cooled to 27° C., and then admixed with ampicillin to give aconcentration of 50 μg/ml, and inoculated with 1%(v/v) of the seedculture, followed by culturing at 27° C. for 48 hours under aerationcondition. After collecting cells in the culture by centrifugation,suspending the cells in 10 mM sodium phosphate buffer (pH 7.0),disrupting the cells by ultrasonication, and removing the cell-debris bycentrifugation, supernatant was obtained. About 1,100 units/L-culture ofenzyme activity was detected by the assay of the activity in theresulting supernatant. About 70 ml of enzyme solution containing about61 units/ml of the polypeptide of the present invention, whose specificactivity is about 13.5 units/mg-protein, was obtained by purifying thesupernatant according to the method described in Experiment 1.

EXAMPLE 2

[0276] Production of the Polypeptide of the Present Invention

[0277] A liquid medium containing 5 g/L of “PINE-DEX #4”, a partialstarch hydrolyzate, 20 g/L of polypeptone, 20 g/L of yeast extract, 1g/L of sodium phosphate, and water was placed in a 500-ml Erlenmeyerflask in an amount of 100 ml, sterilized at 121° C. for 15 minutes, andcooled. Then, the liquid medium was sterilely set to pH 7.0, and admixedwith ampicillin sodium salt to give a final concentration of 100 μg/ml.A transformant, BGN2, obtained by the method in Experiment 6-2, wasinoculated into the above liquid medium, and cultured at 27° C. and at230 rpm for 24 hours to obtain the seed culture. Subsequently, about 18L of a fresh preparation of the same liquid culture medium as used aboveseed culture was placed in a 30-L ferment6r, sterilized with the samemanner, cooled to 27° C., and then admixed with ampicillin to give aconcentration of 50 μg/ml, and inoculated with 1%(v/v) of the seedculture, followed by culturing at 27° C. for 48 hours under aerationcondition. Culture supernatant was obtained by centrifuging theresultant culture. About 750 units/L-culture of enzyme activity wasdetected by the assay of the activity in the resulting culturesupernatant. About 75 ml of enzyme solution containing about 72 units/mlof the polypeptide of the present invention, whose specific activity isabout 12.6 units/mg-protein, was obtained by purifying the supernatantaccording to the method described in Experiment 2.

EXAMPLE 3

[0278] Production of a Syrupy Composition ContainingCyclotetrasaccharide

[0279] A tapioca starch was prepared into an about 25% starchsuspension, admixed with 0.2%/g-starch, d.s.b., of “NEOSPITASE”, anα-amylase commercialized by Nagase Biochemicals, Ltd., Kyoto, Japan, andthen heated at 85-90° C. for about 25 minutes. After autoclaving at 120°C. for 20 minutes, the reaction mixture was cooled to about 35° C. toobtain a liquefied-solution with a DE about four. The liquefied solutionwas admixed with2.2 units/g-starch,d.s.b., of the polypeptide of thepresent invention, obtained in Example 1, 6.6 units/g-starch, d.s.b., ofα-isomaltosyl-transferring enzyme obtained by the method in Experiment1-4, and 10 units/g-starch, d.s.b., of cyclomaltodextringlucanotransferase commercialized by Hayashibara BiochemicalLaboratories Inc., followed by the enzymatic reaction at pH 6.0 and at35° C. for 48 hours. After heating to 95° C. for 30 minutes, thereaction mixture was adjusted at pH 5.0 and 50° C., admixed with 300units/g-starch, d.s.b., of “TRANSGLUCOSIDASE L AMANO™”, an α-glucosidasecommercialized by Amano Pharmaceutical Co., Ltd., Aichi, Japan, followedby the enzymatic reaction for 24 hours. Further the reaction mixture wasmixed with 30 units/g-starch, d.s.b., of “GLUCOZYME”, a glucoamylasepreparation commercialized by Nagase Biochemicals, Ltd., Kyoto, Japan,and then enzymatically reacted for 17 hours. The reaction mixture washeated to 95° C. and kept for 30 minute, and then cooled and filtered toobtain a filtrate. According to the conventional manner, the resultingfiltrate was decolored with activated charcoal, desalted and purifiedwith ion exchangers in H— and OH— forms, and then concentrated into a60% cyclotetrasaccharide syrup in a yield of about 90% to the materialstarch, d.s.b.

[0280] Since the product contains, on a dry solid basis, 36.4% glucose,58.1% cyclotetrasaccharide, and 3.5% of other saccharides and has a mildsweetness, an adequate viscosity, moisture-retaining ability,clathrating ability, it can be advantageously used in a variety ofcompositions such as food products, cosmetics, and pharmaceuticals as asweetener, taste-improving agent, quality-improving agent,syneresis-preventing agent, stabilizer, discoloration-preventing agent,filler, and clathrating agent.

EXAMPLE 4

[0281] Production of a Crystalline Powder of Cyclotetrasaccharide

[0282] A corn starch was prepared into a 25% starch suspension, admixedwith calcium carbonate to give a final concentration of 0.1%, adjustedto pH 6.5, and admixed with 0.3%/g-starch of “THERMAMYL 60 L”, anα-amylase commercialized by Novo Industries A/S, Copenhagen, Denmark,and then heated at 95° C. for 15 minutes. After autoclaving at 120° C.for 20 minutes, the reaction mixture was cooled to 35° C. to obtain aliquefied solution with a DE of about four. To the liquefied solutionwas added 2.5 units/g-starch, d.s.b., of the polypeptide of the presentinvention, obtained in Example 1, 7.0 units/g-starch, d.s.b., ofα-isomaltosyl-transferring enzyme obtained by the method in Experiment1-4, and 10 units/g-starch, d.s.b., of cyclomaltodextringlucanotransferase commercialized by Hayashibara BiochemicalLaboratories Inc., followed by the enzymatic reaction at pH 6.0 and 35°C. for 48 hours. After heating to 95° C. for 30 minutes, the reactionmixture was adjusted at pH 5.0, and 50° C., admixed with 300units/g-starch, d.s.b., of “TRANSGLUCOSIDASE L AMANO™”, an α-glucosidasecommercialized by Amano Pharmaceutical Co., Ltd., Aichi, Japan, followedby the enzymatic reaction for 24 hours. Further the reaction mixture wasmixed with 30 units/g-starch, d.s.b., of “GLUCOZYME”, a glucoamylasepreparation commercialized by Nagase Biochemicals, Ltd., Kyoto, Japan,and then enzymatically reacted for 24 hours. The reaction mixture washeated to 95° C. and kept for 30 minute, and then cooled and filtered toobtain a filtrate. According to the conventional manner, the resultingfiltrate was decolored with activated charcoal, desalted and purifiedwith ion exchangers in H— and OH— forms, and then concentrated into a60% syrup containing, on a dry solid basis, 34.2% glucose, 62.7%cyclotetrasaccharide, and 3.1% of other saccharides.

[0283] The resulting saccharide solution was subjected to a columnchromatography using “AMBERLITE CR-1310 (Na-form)”, a strong acidcation-exchanger resin commercialized by Japan Organo Co., Ltd., Tokyo,Japan. The resin was packed into four-jacketed stainless steel columnshaving a diameter of 5.4 cm, which were then cascaded in series to givea total gel bed depth of 20 m. Under the conditions of keeping the innercolumn temperature at 60° C., the saccharide solution was fed to thecolumns in a volume of 5%(v/v) and fractionated by feeding to thecolumns hot water heated to 60° C. at an SV (space velocity) of 0.13 toobtain high cyclotetrasaccharide content fractions while monitoring thesaccharide composition of eluate by HPLC, and then collected the highcyclotetrasaccharide content solution containing about 98%, d.s.b., ofcyclotetrasaccharide.

[0284] The solution was concentrated to give a concentration of about70% and then placed in a crystallizer, admixed with about 2% crystallinecyclotetrasaccharide penta- or hexa-hydrate as seed crystal, andgradually cooled to obtain a massecuite with a crystallinity of about45%. The massecuite was sprayed from a nozzle equipped on top of dryingtower at high pressure of 150 kg/cm². Simultaneously, hot air heated to85° C. was being brown down from the upper part of the drying tower, andthe resulting crystal powder was collected on a transporting wireconveyor provided on the basement of the tower and gradually moved outof the tower while blowing thereunto a hot air heated to 45° C. Theresulting crystalline powder was injected to an aging tower and aged for10 hours while a hot air was being blown to the contents to completecrystallization and drying to obtain a crystalline powder ofcyclotetrasaccharide penta- or hexa-hydrate in a yield of about 20% tothe material starch, d.s.b.

[0285] Since the product has a relatively low reducibility, doessubstantially neither cause the amino-carbonyl reaction nor exhibithygroscopicity, and has a satisfactory handleability, mild lowsweetness, adequate viscosity, moisture-retaining ability, clathratingability, and substantially non-digestibility, it can be advantageouslyused in a variety of compositions such as food products, cosmetics, andpharmaceuticals as a sweetener, low calorie food, taste-improving agent,flavor-improving agent, quality-improving agent, syneresis-preventingagent, stabilizer, filler, clathrating agent, and base forpulverization.

EXAMPLE 5

[0286] Production of a Crystalline Powder of Cyclotetrasaccharide

[0287] A corn starch was prepared into a 30% starch suspension, admixedwith calcium carbonate to give a concentration of 0.1%, adjusted to pH6.5, and admixed with 0.3%/g-starch, d.s.b., of “THERMAMYL 60 L”, anα-amylase commercialized by Novo Industries A/S, Copenhagen, Denmark,and then heated at 95° C. for 15 minutes. After autoclaving at 120° C.for 20 minutes, the reaction mixture was cooled to 51° C. to obtain aliquefied solution with a DE of about four. To the liquefied solutionwas added 2.4 units/g-starch, d.s.b., of the polypeptide of the presentinvention, obtained in Example 2, 8.0 units/g-starch, d.s.b., ofα-isomaltosyl-transferring enzyme obtained by the method in Experiment2-4, and 3 units/g-starch, d.s.b., of cyclomaltodextringlucanotransferase commercialized by Hayashibara BiochemicalLaboratories Inc., followed by the enzymatic reaction at pH 5.5 and 51°C. for 48 hours. After heating to 95° C. for 30 minutes, the reactionmixture was adjusted to pH 5.0, and 50° C., admixed with 300units/g-starch of “TRANSGLUCOSIDASE L AMANOυ”, an α-glucosidasecommercialized by Amano Pharmaceutical Co., Ltd., Aichi, Japan, followedby the enzymatic reaction for 24 hours. Further the reactionmixturewasmixed with 30 units/g-starch, d.s.b., of “GLUCOZYME”, aglucoamylase preparation commercialized by Nagase Biochemicals, Ltd.,Kyoto, Japan, and then reacted for 17 hours. The reaction mixture washeated to 95° C. and kept for 30 minute, and then cooled and filtered toobtain a filtrate. According to the conventional manner, the resultingfiltrate was decolored with activated charcoal, desalted and purifiedwith ion exchangers in H— and OH— forms, and then concentrated into a60% syrup containing, on a dry solid basis, 46.8% glucose, 44.0%cyclotetrasaccharide, and 9.8% of other saccharides. In order toincrease the content of cyclotetrasaccharide, the resulting saccharidesyrup was fractionated by a column chromatography using a strong acidcation-exchanger resin described in Example 5, and then collected thehigh cyclotetrasaccharide content fractions. After purifying,concentrating, and spray-drying, a powdery product containingcyclotetrasaccharide was obtained in a yield of about 45% to thematerial starch, d.s.b.

[0288] Since the product contains, on a dry solid basis, 3.7% glucose,80.5% cyclotetrasaccharide, and 15.8% of other saccharides and has amild sweetness, an adequate viscosity, moisture-retaining ability,clathrating ability, it can be advantageously used in a variety ofcompositions such as food products, cosmetics, and pharmaceuticals as asweetener, taste-improving agent, quality-improving agent,syneresis-preventing agent, stabilizer, discoloration-preventing agent,filler, clathrating agent, and base for pulverization.

[0289] The present invention, having these outstanding functions andeffects, is a significantly important invention that greatly contributesto this art.

1 32 1 1249 PRT Bacillus globisporus C11 1 Tyr Val Ser Ser Leu Gly AsnLeu Ile Ser Ser Ser Val Thr Gly Asp 1 5 10 15 Thr Leu Thr Leu Thr ValAsp Asn Gly Ala Glu Pro Ser Asp Asp Leu 20 25 30 Leu Ile Val Gln Ala ValGln Asn Gly Ile Leu Lys Val Asp Tyr Arg 35 40 45 Pro Asn Ser Ile Thr ProSer Ala Lys Thr Pro Met Leu Asp Pro Asn 50 55 60 Lys Thr Trp Ser Ala ValGly Ala Thr Ile Asn Thr Thr Ala Asn Pro 65 70 75 80 Met Thr Ile Thr ThrSer Asn Met Lys Ile Glu Ile Thr Lys Asn Pro 85 90 95 Val Arg Met Thr ValLys Lys Ala Asp Gly Thr Thr Leu Phe Trp Glu 100 105 110 Pro Ser Gly GlyGly Val Phe Ser Asp Gly Val Arg Phe Leu His Ala 115 120 125 Thr Gly AspAsn Met Tyr Gly Ile Arg Ser Phe Asn Ala Phe Asp Ser 130 135 140 Gly GlyAsp Leu Leu Arg Asn Ser Ser Asn His Ala Ala His Ala Gly 145 150 155 160Glu Gln Gly Asp Ser Gly Gly Pro Leu Ile Trp Ser Thr Ala Gly Tyr 165 170175 Gly Leu Leu Val Asp Ser Asp Gly Gly Tyr Pro Tyr Thr Asp Ser Thr 180185 190 Thr Gly Gln Met Glu Phe Tyr Tyr Gly Gly Thr Pro Pro Glu Gly Arg195 200 205 Arg Tyr Ala Lys Gln Asn Val Glu Tyr Tyr Ile Met Leu Gly ThrPro 210 215 220 Lys Glu Ile Met Thr Asp Val Gly Glu Ile Thr Gly Lys ProPro Met 225 230 235 240 Leu Pro Lys Trp Ser Leu Gly Phe Met Asn Phe GluTrp Asp Thr Asn 245 250 255 Gln Thr Glu Phe Thr Asn Asn Val Asp Thr TyrArg Ala Lys Asn Ile 260 265 270 Pro Ile Asp Ala Tyr Ala Phe Asp Tyr AspTrp Lys Lys Tyr Gly Glu 275 280 285 Thr Asn Tyr Gly Glu Phe Ala Trp AsnThr Thr Asn Phe Pro Ser Ala 290 295 300 Ser Thr Thr Ser Leu Lys Ser ThrMet Asp Ala Lys Gly Ile Lys Met 305 310 315 320 Ile Gly Ile Thr Lys ProArg Ile Val Thr Lys Asp Ala Ser Ala Asn 325 330 335 Val Thr Thr Gln GlyThr Asp Ala Thr Asn Gly Gly Tyr Phe Tyr Pro 340 345 350 Gly His Asn GluTyr Gln Asp Tyr Phe Ile Pro Val Thr Val Arg Ser 355 360 365 Ile Asp ProTyr Asn Ala Asn Glu Arg Ala Trp Phe Trp Asn His Ser 370 375 380 Thr AspAla Leu Asn Lys Gly Ile Val Gly Trp Trp Asn Asp Glu Thr 385 390 395 400Asp Lys Val Ser Ser Gly Gly Ala Leu Tyr Trp Phe Gly Asn Phe Thr 405 410415 Thr Gly His Met Ser Gln Thr Met Tyr Glu Gly Gly Arg Ala Tyr Thr 420425 430 Ser Gly Ala Gln Arg Val Trp Gln Thr Ala Arg Thr Phe Tyr Pro Gly435 440 445 Ala Gln Arg Tyr Ala Thr Thr Leu Trp Ser Gly Asp Ile Gly IleGln 450 455 460 Tyr Asn Lys Gly Glu Arg Ile Asn Trp Ala Ala Gly Met GlnGlu Gln 465 470 475 480 Arg Ala Val Met Leu Ser Ser Val Asn Asn Gly GlnVal Lys Trp Gly 485 490 495 Met Asp Thr Gly Gly Phe Asn Gln Gln Asp GlyThr Thr Asn Asn Pro 500 505 510 Asn Pro Asp Leu Tyr Ala Arg Trp Met GlnPhe Ser Ala Leu Thr Pro 515 520 525 Val Phe Arg Val His Gly Asn Asn HisGln Gln Arg Gln Pro Trp Tyr 530 535 540 Phe Gly Ser Thr Ala Glu Glu AlaSer Lys Glu Ala Ile Gln Leu Arg 545 550 555 560 Tyr Ser Leu Ile Pro TyrMet Tyr Ala Tyr Glu Arg Ser Ala Tyr Glu 565 570 575 Asn Gly Asn Gly LeuVal Arg Pro Leu Met Gln Ala Tyr Pro Thr Asp 580 585 590 Ala Ala Val LysAsn Tyr Thr Asp Ala Trp Met Phe Gly Asp Trp Leu 595 600 605 Leu Ala AlaPro Val Val Asp Lys Gln Gln Thr Ser Lys Asp Ile Tyr 610 615 620 Leu ProSer Gly Ser Trp Ile Asp Tyr Ala Arg Gly Asn Ala Ile Thr 625 630 635 640Gly Gly Gln Thr Ile Arg Tyr Ser Val Asn Pro Asp Thr Leu Thr Asp 645 650655 Met Pro Leu Phe Ile Lys Lys Gly Ala Ile Ile Pro Thr Gln Lys Val 660665 670 Gln Asp Tyr Val Gly Gln Ala Ser Val Thr Ser Val Asp Val Asp Val675 680 685 Phe Pro Asp Thr Thr Gln Ser Ser Phe Thr Tyr Tyr Asp Asp AspGly 690 695 700 Ala Ser Tyr Asn Tyr Glu Ser Gly Thr Tyr Phe Lys Gln AsnMet Thr 705 710 715 720 Ala Gln Asp Asn Gly Ser Gly Ser Leu Ser Phe ThrLeu Gly Ala Lys 725 730 735 Ser Gly Ser Tyr Thr Pro Ala Leu Gln Ser TyrIle Val Lys Leu His 740 745 750 Gly Ser Ala Gly Thr Ser Val Thr Asn AsnSer Ala Ala Met Thr Ser 755 760 765 Tyr Ala Ser Leu Glu Ala Leu Lys AlaAla Ala Gly Glu Gly Trp Ala 770 775 780 Thr Gly Lys Asp Ile Tyr Gly AspVal Thr Tyr Val Lys Val Thr Ala 785 790 795 800 Gly Thr Ala Ser Ser LysSer Ile Ala Val Thr Gly Val Ala Ala Val 805 810 815 Ser Ala Thr Thr SerGln Tyr Glu Ala Glu Asp Ala Ser Leu Ser Gly 820 825 830 Asn Ser Val AlaAla Lys Ala Ser Ile Asn Thr Asn His Thr Gly Tyr 835 840 845 Thr Gly ThrGly Phe Val Asp Gly Leu Gly Asn Asp Gly Ala Gly Val 850 855 860 Thr PheTyr Pro Lys Val Lys Thr Gly Gly Asp Tyr Asn Val Ser Leu 865 870 875 880Arg Tyr Ala Asn Ala Ser Gly Thr Ala Lys Ser Val Ser Ile Phe Val 885 890895 Asn Gly Lys Arg Val Lys Ser Thr Ser Leu Ala Asn Leu Ala Asn Trp 900905 910 Asp Thr Trp Ser Thr Gln Ser Glu Thr Leu Pro Leu Thr Ala Gly Val915 920 925 Asn Val Val Thr Tyr Lys Tyr Tyr Ser Asp Ala Gly Asp Thr GlyAsn 930 935 940 Val Asn Ile Asp Asn Ile Thr Val Pro Phe Ala Pro Ile IleGly Lys 945 950 955 960 Tyr Glu Ala Glu Ser Ala Glu Leu Ser Gly Gly SerSer Leu Asn Thr 965 970 975 Asn His Trp Tyr Tyr Ser Gly Thr Ala Phe ValAsp Gly Leu Ser Ala 980 985 990 Val Gly Ala Gln Val Lys Tyr Asn Val AsnVal Pro Ser Ala Gly Ser 995 1000 1005 Tyr Gln Val Ala Leu Arg Tyr AlaAsn Gly Ser Ala Ala Thr Lys 1010 1015 1020 Thr Leu Ser Thr Tyr Ile AsnGly Ala Lys Leu Gly Gln Thr Ser 1025 1030 1035 Phe Thr Ser Pro Gly ThrAsn Trp Asn Val Trp Gln Asp Asn Val 1040 1045 1050 Gln Thr Val Thr LeuAsn Ala Gly Ala Asn Thr Ile Ala Phe Lys 1055 1060 1065 Tyr Asp Ala AlaAsp Ser Gly Asn Ile Asn Val Asp Arg Leu Leu 1070 1075 1080 Leu Ser ThrSer Ala Ala Gly Thr Pro Val Ser Glu Gln Asn Leu 1085 1090 1095 Leu AspAsn Pro Gly Phe Glu Arg Asp Thr Ser Gln Thr Asn Asn 1100 1105 1110 TrpIle Glu Trp His Pro Gly Thr Gln Ala Val Ala Phe Gly Val 1115 1120 1125Asp Ser Gly Ser Thr Thr Asn Pro Pro Glu Ser Pro Trp Ser Gly 1130 11351140 Asp Lys Arg Ala Tyr Phe Phe Ala Ala Gly Ala Tyr Gln Gln Ser 11451150 1155 Ile His Gln Thr Ile Ser Val Pro Val Asn Asn Val Lys Tyr Lys1160 1165 1170 Phe Glu Ala Trp Val Arg Met Lys Asn Thr Thr Pro Thr ThrAla 1175 1180 1185 Arg Ala Glu Ile Gln Asn Tyr Gly Gly Ser Ala Ile TyrAla Asn 1190 1195 1200 Ile Ser Asn Ser Gly Val Trp Lys Tyr Ile Ser ValSer Asp Ile 1205 1210 1215 Met Val Thr Asn Gly Gln Ile Asp Val Gly PheTyr Val Asp Ser 1220 1225 1230 Pro Gly Gly Thr Thr Leu His Ile Asp AspVal Arg Val Thr Lys 1235 1240 1245 Gln 2 1251 PRT Bacillus globisporusN75 2 His Val Ser Ala Leu Gly Asn Leu Leu Ser Ser Ala Val Thr Gly Asp 15 10 15 Thr Leu Thr Leu Thr Ile Asp Asn Gly Ala Glu Pro Asn Asp Asp Ile20 25 30 Leu Val Leu Gln Ala Val Gln Asn Gly Ile Leu Lys Val Asp Tyr Arg35 40 45 Pro Asn Gly Val Ala Pro Ser Ala Asp Thr Pro Met Leu Asp Pro Asn50 55 60 Lys Thr Trp Pro Ser Ile Gly Ala Val Ile Asn Thr Ala Ser Asn Pro65 70 75 80 Met Thr Ile Thr Thr Pro Ala Met Lys Ile Glu Ile Ala Lys AsnPro 85 90 95 Val Arg Leu Thr Val Lys Lys Pro Asp Gly Thr Ala Leu Leu TrpGlu 100 105 110 Pro Pro Thr Gly Gly Val Phe Ser Asp Gly Val Arg Phe LeuHis Gly 115 120 125 Thr Gly Asp Asn Met Tyr Gly Ile Arg Ser Phe Asn AlaPhe Asp Ser 130 135 140 Gly Gly Asp Leu Leu Arg Asn Ser Ser Thr Gln AlaAla Arg Ala Gly 145 150 155 160 Asp Gln Gly Asn Ser Gly Gly Pro Leu IleTrp Ser Thr Ala Gly Tyr 165 170 175 Gly Val Leu Val Asp Ser Asp Gly GlyTyr Pro Phe Thr Asp Glu Ala 180 185 190 Thr Gly Lys Leu Glu Phe Tyr TyrGly Gly Thr Pro Pro Glu Gly Arg 195 200 205 Arg Tyr Thr Lys Gln Asp ValGlu Tyr Tyr Ile Met Leu Gly Thr Pro 210 215 220 Lys Glu Ile Met Ser GlyVal Gly Glu Ile Thr Gly Lys Pro Pro Met 225 230 235 240 Leu Pro Lys TrpSer Leu Gly Phe Met Asn Phe Glu Trp Asp Leu Asn 245 250 255 Glu Ala GluLeu Lys Asn His Val Asp Thr Tyr Arg Ala Lys Asn Ile 260 265 270 Pro IleAsp Gly Tyr Ala Ile Asp Phe Asp Trp Lys Lys Tyr Gly Glu 275 280 285 AsnAsn Tyr Gly Glu Phe Ala Trp Asn Thr Ala Asn Phe Pro Ser Ala 290 295 300Ala Thr Thr Ala Leu Lys Ser Gln Met Asp Ala Lys Gly Ile Lys Met 305 310315 320 Ile Gly Ile Thr Lys Pro Arg Ile Ala Thr Lys Asp Phe Ser Asn Asn325 330 335 Pro Thr Val Gln Gly Thr Asp Ala Ala Ser Gly Gly Tyr Phe TyrPro 340 345 350 Gly His Ser Glu Tyr Lys Asp Tyr Phe Ile Pro Val Phe ValArg Ser 355 360 365 Ile Asp Pro Tyr Asn Pro Ala Ala Arg Ser Trp Phe TrpAsn His Ser 370 375 380 Lys Asp Ala Phe Asp Lys Gly Ile Val Gly Trp TrpAsn Asp Glu Thr 385 390 395 400 Asp Ala Val Ser Ser Gly Gly Ala Ser TyrTrp Phe Gly Asn Phe Thr 405 410 415 Thr Gly His Met Ser Gln Ala Leu TyrGlu Gly Gln Arg Ala Tyr Thr 420 425 430 Ser Asn Ala Gln Arg Val Trp GlnThr Ala Arg Thr Phe Tyr Pro Gly 435 440 445 Ala Gln Arg Tyr Ala Thr ThrLeu Trp Ser Gly Asp Ile Gly Ile Gln 450 455 460 Tyr Thr Lys Gly Glu ArgIle Asn Trp Ala Ala Gly Met Gln Glu Gln 465 470 475 480 Arg Ala Val MetLeu Ser Ser Ile Asn Asn Gly Gln Val Lys Trp Gly 485 490 495 Met Asp ThrGly Gly Phe Asn Gln Gln Asp Gly Thr Thr Asn Asn Pro 500 505 510 Asn ProAsp Leu Tyr Ala Arg Trp Met Gln Phe Ser Ala Leu Thr Pro 515 520 525 ValPhe Arg Val His Gly Asn Asn His Gln Gln Arg Gln Pro Trp Tyr 530 535 540Tyr Gly Ser Thr Ala Glu Glu Ala Ser Lys Glu Ala Leu Gln Leu Arg 545 550555 560 Tyr Ser Leu Ile Pro Tyr Met Tyr Ala Tyr Glu Arg Ser Ala Tyr Glu565 570 575 Asn Gly Asn Gly Leu Val Arg Pro Leu Met Gln Glu Tyr Pro AlaAsp 580 585 590 Ala Asn Ala Lys Asn Tyr Leu Asp Ala Trp Met Phe Gly AspTrp Leu 595 600 605 Leu Ala Ala Pro Val Val Glu Lys Gln Gln Thr Ser LysGlu Ile Tyr 610 615 620 Leu Pro Ala Gly Thr Trp Ile Asp Tyr Asn Arg GlyThr Val Leu Thr 625 630 635 640 Gly Gly Gln Lys Ile Ser Tyr Ala Val AsnPro Asp Thr Leu Thr Asp 645 650 655 Ile Pro Leu Phe Ile Lys Lys Gly AlaIle Ile Pro Ser Gln Lys Val 660 665 670 Gln Asp Tyr Val Gly Gln Ala ProVal Gln Thr Val Asp Val Asp Val 675 680 685 Phe Pro Asn Thr Ala Gln SerSer Phe Thr Tyr Tyr Asp Asp Asp Gly 690 695 700 Ser Ser Tyr Asn Tyr GluSer Gly Ala Tyr Phe Lys Gln Leu Met Thr 705 710 715 720 Ala Gln Asp AsnGly Ser Gly Ala Leu Ser Phe Thr Leu Gly Ala Lys 725 730 735 Thr Gly ThrTyr Ser Pro Ala Leu Gln Ser Tyr Ile Val Lys Leu His 740 745 750 Gly AlaAla Gly Ala Ser Val Thr Ser Asn Gly Ala Ala Leu Ala Ser 755 760 765 TyrAla Ser Leu Gln Ala Leu Lys Ala Ser Ala Ser Glu Gly Trp Ala 770 775 780Lys Gly Lys Asp Ile Tyr Gly Asp Val Thr Tyr Val Lys Leu Ser Ala 785 790795 800 Gly Ala Ala Ala Ala Lys Ala Ile Ala Val Thr Gly Asn Ser Pro Val805 810 815 Ser Val Ala Asp Val Gln Tyr Glu Ala Glu Glu Ala Ser Leu SerGly 820 825 830 Asn Thr Thr Ala Thr Lys Ala Thr Val Asn Thr Asn His AlaGly Tyr 835 840 845 Thr Gly Ser Gly Phe Val Asp Gly Leu Ser Asn Pro GlyAla Ala Val 850 855 860 Thr Phe Tyr Pro Lys Val Lys Thr Gly Gly Asp TyrAsn Val Ser Leu 865 870 875 880 Arg Tyr Ala Asn Ser Thr Gly Ala Ala LysSer Val Ser Ile Phe Val 885 890 895 Asn Gly Lys Arg Val Lys Ser Thr SerLeu Ala Asn Leu Pro Asn Trp 900 905 910 Asp Thr Trp Gly Thr Gln Ala GluThr Leu Pro Leu Thr Ala Gly Thr 915 920 925 Asn Val Val Thr Tyr Lys PheTyr Ser Asp Ala Gly Asp Thr Gly Ser 930 935 940 Val Asn Leu Asp Asn IleThr Val Pro Phe Ala Pro Ala Ile Gly Lys 945 950 955 960 Tyr Glu Ala GluSer Ala Glu Leu Ser Gly Gly Ser Thr Val Asn Gln 965 970 975 Asn His TrpPhe Tyr Ser Gly Thr Ala Phe Val Asp Gly Leu Thr Ala 980 985 990 Pro GlyAla Gln Val Lys Tyr Thr Val Asn Ala Pro Ala Ala Gly Ser 995 1000 1005Tyr Gln Ile Ala Leu Arg Tyr Ala Asn Gly Thr Gly Ala Ala Lys 1010 10151020 Thr Leu Ser Thr Tyr Val Asn Gly Thr Lys Leu Gly Gln Thr Ala 10251030 1035 Phe Ala Ser Pro Gly Gly Asn Trp Asn Val Trp Gln Asp Ser Val1040 1045 1050 Gln Thr Val Ala Leu Ala Ala Gly Thr Asn Thr Ile Ala PheLys 1055 1060 1065 Tyr Asp Ala Gly Asp Ser Gly Ser Gly Ser Val Asn LeuAsp Arg 1070 1075 1080 Leu Leu Leu Ser Ala Ala Ala Pro Gly Val Pro ValSer Glu Gln 1085 1090 1095 Asn Leu Leu Asp Asn Gly Gly Phe Glu Arg AspPro Ser Gln Ser 1100 1105 1110 Ser Asn Trp Thr Glu Trp His Pro Ala SerGln Ala Ile Ala Tyr 1115 1120 1125 Gly Ile Asp Ser Gly Ser Gly Met AsnPro Pro Glu Ser Pro Trp 1130 1135 1140 Ala Gly Asp Lys Arg Ala Tyr PheTyr Ala Ala Gly Pro Tyr Gln 1145 1150 1155 Gln Ser Ile His Gln Thr ValSer Val Pro Val Asn Asn Ala Lys 1160 1165 1170 Tyr Lys Phe Glu Ala TrpVal Leu Leu Lys Asn Thr Thr Pro Thr 1175 1180 1185 Thr Ala Arg Val GluIle Gln Asn Tyr Gly Gly Ser Pro Ile Phe 1190 1195 1200 Thr Asn Ile SerLys Asp Gly Val Trp Lys Tyr Ile Ser Val Ser 1205 1210 1215 Asp Ile GlnVal Thr Asn Gly Gln Ile Asp Ile Gly Phe Tyr Val 1220 1225 1230 Asp SerPro Gly Gly Thr Thr Leu His Ile Asp Asp Val Arg Val 1235 1240 1245 ThrLys Gln 1250 3 929 PRT Arthrobacter globiformis A19 3 Ala Pro Leu GlyVal Gln Arg Ala Gln Phe Gln Ser Gly Ser Ser Tyr 1 5 10 15 Leu Val ValGlu Val Leu Asp Asp Asp Leu Val His Phe Glu Leu Ala 20 25 30 Gly Gly GlyThr Ala Pro Gly Thr Gly Ser Pro Leu Phe Thr Thr Pro 35 40 45 Gln Val AlaLys His Asp Tyr Ala Gly Pro Asp Val Phe Thr Gln Thr 50 55 60 Gly Ser ValLeu Gln Thr Ala Ala Met Arg Ile Glu Val Asp Pro Ala 65 70 75 80 Asp LeuCys Val Thr Ala Thr Asp Ile Thr Arg Thr Pro Asn Leu Val 85 90 95 Leu HisGlu Ala Cys Pro Ala Asp Leu Gly Gln Ala Trp Lys Gly Leu 100 105 110 AsnIle Thr Arg Ser Ala Met Glu Asn Ala Tyr Gly Leu Gly Gln Gln 115 120 125Phe Phe Thr Gly Gly Ser Ala Asp Gly Asp Trp Val Gly Arg Thr Arg 130 135140 Thr Pro Gly Gly Thr Tyr Gly Asn Ala Met Val Phe Asp Pro Glu Asn 145150 155 160 Gly Pro Val Gly Asn Thr Gln Ile Pro Val Leu Phe Ala Val GlyAsp 165 170 175 Asp Asn Ala Asn Tyr Gly Leu Phe Val Asp Gln Leu Tyr LysGln Glu 180 185 190 Trp Asn Leu Thr Gly Asp Pro Trp Thr Val Arg Met TrpGly Asp Gln 195 200 205 Val Arg Trp Tyr Leu Met Ser Gly Asp Asp Leu ProAsp Leu Arg His 210 215 220 Asp Tyr Met Glu Leu Thr Gly Thr Pro Pro ValPro Pro Lys Lys Ala 225 230 235 240 Phe Gly Leu Trp Val Ser Glu Phe GlyTyr Asp Asn Trp Ser Glu Val 245 250 255 Asp Asn Thr Ile Ala Gly Leu ArgSer Ala Asp Phe Pro Val Asp Gly 260 265 270 Ala Met Leu Asp Val Gln TrpPhe Gly Gly Val Thr Ala Asp Ser Asp 275 280 285 Asp Thr Arg Met Gly ThrLeu Asp Trp Asp Thr Ser Arg Phe Pro Asp 290 295 300 Pro Ala Gly Lys IleAla Asp Leu Ala Glu Asp Gly Val Gly Ile Ile 305 310 315 320 Pro Ile GluGlu Ser Tyr Val Gly Arg Asn Leu Pro Glu His Ala Arg 325 330 335 Met AlaAla Asp Gly Tyr Leu Val Arg Ser Gly Cys Ala Thr Cys Pro 340 345 350 ProVal Tyr Leu Thr Gly Asn Pro Trp Trp Gly Lys Gly Gly Met Ile 355 360 365Asp Trp Thr Gln Pro Glu Ala Gly Ala Val Trp His Asp Glu Gln Arg 370 375380 Gln His Leu Val Asp Glu Gly Val Leu Gly His Trp Leu Asp Leu Gly 385390 395 400 Glu Pro Glu Met Tyr Asp Pro Asn Asp Trp Thr Ala Gly Val IlePro 405 410 415 Gly Lys His Ala His Ala Asp Tyr His Asn Ala Tyr Asn LeuLeu Trp 420 425 430 Ala Gln Ser Ile Ala Asp Gly Tyr Ala Asp Asn Gly ValGln Lys Arg 435 440 445 Pro Phe Met Leu Thr Arg Ala Ala Ala Ala Gly IleGln Arg His Gly 450 455 460 Ala Gly Met Trp Ser Ala Asp Ile Gly Ser ThrMet Lys Ala Leu Gly 465 470 475 480 Ser Gln Gln Asn Ala Gln Met His MetSer Met Ser Gly Ile Asp Tyr 485 490 495 Tyr Gly Ser Asp Ile Gly Gly PheArg Arg Glu Met Ala Asp Gly Asp 500 505 510 Val Asn Glu Leu Tyr Thr GlnTrp Phe Ala Asp Ser Ala Trp Phe Asp 515 520 525 Thr Pro Leu Arg Pro HisThr Asp Asn Leu Cys Asn Cys Leu Glu Thr 530 535 540 Ser Pro Asp Ser IleGly Asp Val Ala Ser Asn Arg Glu Asn Leu Val 545 550 555 560 Arg Arg TyrGlu Leu Ala Pro Tyr Tyr Tyr Ser Leu Ala His Arg Ala 565 570 575 His GlnPhe Gly Glu Pro Leu Ala Pro Pro Leu Val Tyr Tyr Tyr Gln 580 585 590 AsnAsp Asp His Val Arg Glu Met Gly His Gln Lys Met Leu Gly Arg 595 600 605Asp Leu Leu Ile Ala Ile Val Ala Gly Glu Gly Glu Arg Glu Arg Asp 610 615620 Val Tyr Leu Pro Ala Gly Glu Trp Ile Asp Ile His Thr Asn Glu Arg 625630 635 640 Ile Gln Ser Thr Gly Gln Trp Ile Asp Asn Val Pro Leu Trp ArgAsp 645 650 655 Gly Val Phe Thr Leu Pro Ala Tyr Ala Arg Ala Gly Ala IleIle Pro 660 665 670 Lys Ala Phe Val Asp Ala Ser Thr Lys Asp Ile Thr GlyLys Arg Glu 675 680 685 Asp Ala Ala Val Arg Asn Glu Leu Ile Ala Thr ValTyr Ala Asp Asp 690 695 700 Val Ala Ser Asp Phe Thr Leu Tyr Glu Asp AspGly Ala Thr Thr Ala 705 710 715 720 Tyr Ala Asp Gly Ala Val Arg Thr ThrGln Ile Ser Gln Ser Leu Thr 725 730 735 Asn Gly Val Ala Thr Val Thr ValGly Ala Ala Ser Gly Thr Tyr Ser 740 745 750 Gly Ala Pro Ser Thr Arg ProThr Val Val Glu Leu Val Thr Asp Gly 755 760 765 Thr Gln Ala Ser Thr ValSer Leu Gly Ser Val Pro Leu Thr Glu His 770 775 780 Ala Asn Lys Ala AlaPhe Asp Ala Ala Ser Ser Gly Trp Tyr Asn Ala 785 790 795 800 Gly Gly GlyLeu Val Val Ala Lys Ala Ala Ser Ser Ser Val Asn Thr 805 810 815 Ala LysThr Phe Ser Phe Thr Leu Gly Glu Glu Ser Val Trp Ala Thr 820 825 830 PheSer Cys Glu Asn Ala Thr Thr Thr Phe Gly Gln Ser Val Tyr Val 835 840 845Val Gly Asn Val Pro Gln Leu Gly Asn Trp Ser Pro Ala Asp Ala Val 850 855860 Lys Leu Glu Pro Ser Ala Tyr Pro Thr Trp Thr Gly Val Val Arg Asn 865870 875 880 Leu Pro Pro Ser Ser Thr Val Glu Trp Lys Cys Ile Lys Arg GlnGlu 885 890 895 Ala Gly Leu Pro Asn Thr Ala Asp Ala Trp Glu Pro Gly GlyAsn Asn 900 905 910 Ile Leu Ser Thr Pro Pro Ser Gly Ser Ala Gly Ile ThrThr Gly Ala 915 920 925 Phe 4 3747 DNA Bacillus globisporus C11 4tatgtcagca gcctaggaaa tctcatttct tcgagtgtca ccggagatac cttgacgcta 60actgttgata acggtgcgga gccgagtgat gacctcttga ttgttcaagc ggtgcaaaac 120ggtattttga aggtggatta tcgtccaaat agcataacgc cgagcgcgaa gacgccgatg 180ctggatccga acaaaacttg gtcagctgta ggagctacga ttaatacgac agccaatcca 240atgaccatca cgacttccaa tatgaagatt gagattacca agaatccagt acgaatgacg 300gtcaagaagg cggacggcac tacgctattc tgggaaccat caggcggagg ggtattctca 360gacggtgtgc gcttccttca tgccacaggg gataatatgt atggcatccg gagcttcaat 420gcttttgata gcgggggtga cctgctgcgg aattcgtcca atcatgccgc ccatgcgggt 480gaacagggag attccggtgg tccgcttatt tggagtacgg caggatatgg actattagtc 540gatagcgatg gcggctaccc ctatacagat agcacaaccg gtcaaatgga gttttattat 600ggtgggaccc ctcctgaggg acgtcgttat gcgaaacaaa acgtggaata ttatattatg 660ctcggaaccc ccaaggaaat tatgaccgac gtaggggaaa tcacagggaa accgcctatg 720ctgcctaagt ggtcgcttgg attcatgaac tttgagtggg atacgaatca aacggagttt 780acgaataatg tggatacgta tcgtgccaaa aatatcccca tagatgctta cgccttcgac 840tatgactgga aaaagtacgg ggaaaccaac tatggtgaat tcgcgtggaa tacgactaat 900ttcccttctg cgtcaacgac ttctttaaag tcaacaatgg atgctaaagg catcaaaatg 960atcggaatta caaaaccccg catcgttacg aaggatgctt cagcgaatgt gacgacccaa 1020gggacggacg cgacaaatgg cggttatttt tatccaggcc ataacgagta tcaggattat 1080ttcattcccg taactgtgcg tagtatcgat ccttacaatg ctaacgaacg tgcttggttc 1140tggaatcatt ccacagatgc gcttaataaa gggatcgtag gttggtggaa tgacgagacg 1200gataaagtat cttcgggtgg agcgttatat tggtttggca atttcacaac aggccacatg 1260tctcagacga tgtacgaagg ggggcgggct tacacgagtg gagcgcagcg tgtttggcaa 1320acggctagaa ccttctaccc aggtgcccag cggtatgcga ctacgctttg gtctggcgat 1380attggcattc aatacaataa aggcgaacgg atcaattggg ctgccgggat gcaggagcaa 1440agggcagtta tgctatcctc cgtgaacaat ggccaggtga aatggggcat ggataccggc 1500ggattcaatc agcaggatgg cacgacgaac aatccgaatc ccgatttata cgctcggtgg 1560atgcagttca gtgccctaac gcctgttttc cgagtgcatg ggaacaacca tcagcagcgc 1620cagccatggt acttcggatc gactgcggag gaggcctcca aagaggcaat tcagctgcgg 1680tactccctga tcccttatat gtatgcctat gagagaagtg cttacgagaa tgggaatggg 1740ctcgttcggc cattgatgca agcctatcca acagatgcgg ccgtcaaaaa ttacacggat 1800gcttggatgt ttggtgactg gctgctggct gcacctgtgg tagataaaca gcagacgagt 1860aaggatatct atttaccgtc tgggtcatgg attgactatg cgcgaggcaa tgcaataact 1920ggcggtcaaa ccatccgata ttcggttaat ccggacacgt tgacagacat gcctctcttt 1980attaaaaaag gtgccattat tccaacacag aaagtgcagg attacgtagg gcaggcttcc 2040gtcacttccg ttgatgtgga tgtgtttccg gatacgacgc agtcgagttt cacgtactac 2100gatgatgatg gcgccagtta taactatgag agcggcactt attttaagca aaatatgact 2160gctcaggata atgggtcagg ctcgttaagt tttactttag gagcaaagag tggcagttac 2220acgccggctc tccaatccta tatcgttaag ctgcacggtt ctgctggaac ttctgttacg 2280aataacagcg cagctatgac atcttatgca agcttggaag cattaaaagc tgctgctggg 2340gaaggctggg cgactgggaa ggacatttat ggggatgtca cctatgtgaa agtgacggca 2400ggtacagctt cttctaaatc tattgctgtt acaggtgttg ctgccgtgag cgcaactact 2460tcgcaatacg aagctgagga tgcatcgctt tctggcaatt cggttgctgc aaaggcgtcc 2520ataaacacga atcataccgg atatacggga actggatttg tagatggttt ggggaatgat 2580ggcgctggtg tcaccttcta tccaaaggtg aaaactggcg gtgactacaa tgtctccttg 2640cgttatgcga atgcttcagg cacggctaag tcagtcagta tttttgttaa tggaaaaaga 2700gtgaagtcca cctcgctcgc taatctcgca aattgggaca cttggtctac acaatctgag 2760acactgccgt tgacggcagg tgtgaatgtt gtgacctata aatattactc cgatgcggga 2820gatacaggca atgttaacat cgacaacatc acggtacctt ttgcgccaat tatcggtaag 2880tatgaagcag agagtgctga gctttctggt ggcagctcat tgaacacgaa ccattggtac 2940tacagtggta cggcttttgt agacggtttg agtgctgtag gcgcgcaggt gaaatacaac 3000gtgaatgtcc ctagcgcagg aagttatcag gtagcgctgc gatatgcgaa tggcagtgca 3060gcgacgaaaa cgttgagtac ttatatcaat ggagccaagc tggggcaaac cagttttacg 3120agtcctggta cgaattggaa tgtttggcag gataatgtgc aaacggtgac gttaaatgca 3180ggggcaaaca cgattgcgtt taaatacgac gccgctgaca gcgggaacat caacgtagat 3240cgtctgcttc tttcaacttc ggcagcggga acgccggttt ctgagcagaa cctgctagac 3300aatcccggtt tcgagcgtga cacgagtcaa accaataact ggattgagtg gcatccaggc 3360acgcaagctg ttgcttttgg cgttgatagc ggctcaacca ccaatccgcc ggaatccccg 3420tggtcgggtg ataagcgtgc ctacttcttt gcagcaggtg cctatcaaca aagcatccat 3480caaaccatta gtgttcctgt taataatgta aaatacaaat ttgaagcctg ggtccgcatg 3540aagaatacga cgccgacgac ggcaagagcc gaaattcaaa actatggcgg atcagccatt 3600tatgcgaaca taagtaacag cggtgtttgg aaatatatca gcgtaagtga tattatggtg 3660accaatggtc agatagatgt tggattttac gtggattcac ctggtggaac tacgcttcac 3720attgatgatg tgcgcgtaac caaacaa 3747 5 3753 DNA Bacillus globisporus N75 5catgtgagcg cgctgggcaa cctgctttcc tcggcggtga ccggggatac gctcacgctg 60acgatcgata acggcgcgga accgaatgac gatattctag ttctgcaagc agtccagaac 120ggtattctga aggtggacta ccggccgaac ggtgtagctc caagcgcgga tacgccgatg 180ctggatccca ataaaacctg gccgtccata ggcgccgtta tcaatacagc ctctaatccg 240atgacgatca caacgccggc gatgaagatt gagattgcca aaaatccggt gcgcctgacc 300gtgaaaaaac cggacggcac cgctctgtta tgggaacccc cgaccggcgg cgtcttctcg 360gacggcgtcc gtttcttgca cgggacgggc gacaatatgt acggcatccg cagcttcaat 420gcttttgaca gcggcgggga tctgctgcgc aacagctcca cccaagccgc ccgtgcaggc 480gaccagggca actccggcgg cccgctgatc tggagcacag ccgggtacgg ggtgctcgtt 540gacagcgacg gtgggtatcc gttcacggac gaggctaccg gcaagctgga gttctattac 600ggcggcacgc ctccggaagg ccggcgctat acgaagcagg atgtggagta ctacatcatg 660ctcggcacgc cgaaagagat catgtccggc gtcggggaaa ttacgggcaa accgccgatg 720ctgcccaagt ggtccctggg ctttatgaac ttcgagtggg atctgaatga agctgagctc 780aagaaccatg tggatacgta ccgggccaaa aatattccga tcgacggcta tgcgatcgat 840ttcgattgga agaagtacgg cgagaataat tacggcgaat tcgcttggaa tacggccaat 900ttcccttccg ccgccacgac ggcgctgaag tcgcagatgg acgccaaggg cattaaaatg 960atcggcataa ccaagcctcg catcgcgacg aaggattttt cgaacaatcc taccgtgcag 1020ggaacggacg cggcgagcgg cggttatttt tatccgggac atagcgaata caaggactac 1080ttcatcccgg tctttgtgcg cagcatcgac ccttataacc ctgctgcacg ctcctggttc 1140tggaaccact ccaaggatgc gttcgataaa ggcatcgtag gctggtggaa cgacgagacg 1200gatgcggtat cgtcgggagg ggcctcctac tggttcggca attttacgac cggccatatg 1260tcccaggcgc tttacgaggg acagcgggca tatacgtcga acgcccagcg cgtctggcag 1320acagcgcgca cgttctatcc cggggcgcag cgttatgcga cgacgctctg gtcgggagac 1380atcgggattc agtataccaa gggggaaaga atcaactggg ctgccggcat gcaggagcag 1440cgggcggtga tgctttcttc gatcaacaac ggccaggtca aatggggaat ggacacaggc 1500ggcttcaacc agcaggacgg cacgacgaac aatccgaatc cggacctgta cgccagatgg 1560atgcagttca gcgcgctgac tccggtgttc cgcgtgcatg gcaacaatca ccagcagcgc 1620cagccttggt attatggctc gacagccgag gaggcatcca aggaagcgct ccagctccgt 1680tactccctga ttccttatat gtatgcttac gaaagaagcg cctacgagaa cggtaacgga 1740cttgtccggc cgctgatgca ggaataccct gccgatgcca acgccaaaaa ctatctcgat 1800gcctggatgt tcggcgattg gctgctggcg gcgcctgtgg tcgagaagca gcagacctcc 1860aaggaaatct atctccctgc aggcacttgg attgactaca accggggcac ggtgctcacc 1920ggcggccaga agatcagcta cgccgtcaat cccgacacgc tgacggatat tccgctcttc 1980attaagaagg gcgcgattat cccttcgcag aaggtgcagg actacgtggg ccaggctccc 2040gtccaaacgg tggatgtgga tgtattcccg aatacggcac aatcgagctt tacctattat 2100gacgatgacg gcagcagcta caattatgaa agcggagctt acttcaagca attgatgacg 2160gctcaggaca acggatccgg tgcgctgagc tttacgctgg gcgccaaaac cggcacgtac 2220agccccgcac tgcaatccta tatcgtcaag cttcacgggg ccgcaggcgc gtcggtgaca 2280agcaatgggg cggcgctggc ctcctatgcc agcctgcaag cgctgaaagc ctcagccagt 2340gaaggctggg ccaagggcaa ggacatctac ggcgatgtca cgtatgtcaa gctatccgcg 2400ggggcagcgg cggccaaggc gattgccgtc accggcaaca gcccggtcag cgtggcggat 2460gtgcagtacg aagccgaaga agcttcgctg tccggcaata cgacagcaac caaggcgacc 2520gtgaatacga accacgcagg ctacacgggc agcggcttcg tggatggact gagtaatccg 2580ggagcggcgg ttacgttcta tccgaaggtg aaaacgggcg gagactacaa tgtctcgctg 2640cgctacgcta attcgacggg agcggcaaag agcgtcagca tcttcgttaa cggcaagcgc 2700gtcaaatcca cgtcgctggc gaacctgccg aactgggata cgtgggggac gcaggctgag 2760acactgccgc tgacggcggg gacgaacgtt gtcacctaca agttctactc ggatgccgga 2820gatacgggct cggttaacct ggacaacatc acggtgccct tcgctccggc catcggcaaa 2880tacgaggcgg agagcgccga gctgagcggc ggcagcacgg tcaaccagaa tcattggttc 2940tacagcggca cggcatttgt agatggctta accgcaccgg gcgcccaagt caaatatacc 3000gtgaacgccc cggccgcagg cagctaccag atcgcgcttc gctatgcgaa cggcacgggt 3060gctgcgaaga cgctcagcac gtatgtgaac gggacgaagc tggggcaaac ggccttcgcc 3120agccctggcg gcaactggaa cgtgtggcag gacagcgtgc agaccgtcgc gctcgccgcc 3180ggtacgaaca cgatcgcgtt caagtacgat gccggcgaca gcggcagcgg cagcgtcaat 3240ctggaccgtc tgttgctctc tgccgcagcg ccaggcgtgc ccgtgtccga gcagaacctg 3300ctcgataacg ggggctttga acgcgatccg tcgcagagca gcaactggac cgagtggcat 3360ccggcttcgc aggcgattgc ttacggcatc gacagcggct ccgggatgaa tccgcctgaa 3420tcgccatggg caggcgataa gcgcgcctat ttctatgcgg caggcccgta tcagcaaagc 3480atccatcaaa cagtcagcgt gcctgtcaat aatgccaagt acaagttcga agcctgggta 3540ttgctgaaga atacaacacc gacaacggcc cgggtggaga ttcaaaatta cggcggttcg 3600ccgatcttca cgaacatcag taaagacggc gtctggaaat acatcagcgt cagcgatatt 3660caggtcacga acggccaaat cgatattggc ttctatgtgg attcgcccgg aggcaccacg 3720ctccacatcg acgatgtgcg ggtcaccaag caa 3753 6 2787 DNA Arthrobacterglobiformis A19 6 gctcccctgg gcgtgcaacg cgcgcagttc cagtcggggt cgagctacctcgtcgtcgag 60 gtgctcgatg acgacctcgt ccacttcgag ctggccgggg gcggcaccgcccccggcacg 120 ggctccccgc tgttcacgac gcctcaggtc gcgaagcacg actacgcgggacccgacgtg 180 ttcacccaga ccgggtctgt tctgcagacc gcggcgatgc gcatcgaggtcgatcccgcg 240 gatctgtgcg tgacggccac cgacatcacc cgcaccccga accttgtactgcacgaggcg 300 tgtcccgccg acctcggcca ggcgtggaag gggctgaaca tcacgaggtcggcgatggag 360 aacgcctacg gtctcgggca gcagttcttc acgggcggca gcgcggacggcgactgggtg 420 ggccgcaccc gcaccccggg tggcacctac ggcaacgcga tggtgttcgaccccgagaac 480 gggccggtcg gcaacacgca gatcccggtg ctcttcgcgg tcggcgatgacaacgcgaac 540 tacgggctgt tcgtcgatca gctgtacaag caggaatgga acctcaccggcgacccgtgg 600 acggtgcgca tgtggggcga ccaggtgcgc tggtacctca tgagcggcgacgacctgccc 660 gaccttcgcc acgactacat ggagctgacg ggcaccccgc ccgtgccgccgaagaaggcg 720 ttcgggctct gggtgtcgga gttcggctac gacaactgga gcgaggtcgacaatacgatc 780 gcgggcctgc gctcggccga ctttccggtc gatggcgcga tgctcgacgtacagtggttc 840 gggggcgtca ccgccgactc ggacgacacc cgcatgggca ccctcgattgggacacgtcg 900 aggtttcccg accctgcggg aaagatcgcc gacctcgccg aggacggcgtcggcatcatc 960 ccgatcgagg agtcgtacgt cggtcgcaac ctgccggagc acgcccggatggcggcggac 1020 ggttacctcg tgcgctccgg ctgcgctacg tgcccgccgg tgtacctgacggggaacccc 1080 tggtggggca agggcgggat gatcgactgg acgcagccgg aagccggcgccgtctggcac 1140 gacgagcagc gccagcatct cgtcgacgag ggcgtactgg gccactggctcgatctcggc 1200 gaaccggaga tgtacgaccc gaacgactgg accgccggcg tcatccccggcaagcacgcg 1260 cacgccgact atcacaacgc gtacaacctg ctgtgggcgc agagcatcgccgacgggtac 1320 gccgacaacg gcgtgcagaa gcgtcccttc atgctgacgc gcgccgcggccgccggcatc 1380 cagcgtcatg gcgcgggcat gtggtcagcc gacatcgggt cgaccatgaaggcgctcggg 1440 agccagcaga acgcgcagat gcacatgtcg atgtcgggga tcgactattacggctccgac 1500 atcggcgggt tccggcggga gatggccgac ggcgacgtga acgagctctacacccagtgg 1560 ttcgccgaca gcgcgtggtt cgacactccg ctccggccgc acaccgacaatctctgcaac 1620 tgcctcgaga cgagccccga ctcgatcggc gacgtcgcga gcaaccgcgagaacctggtg 1680 cgccgctacg agctggctcc gtactactac tcgctcgcgc accgcgctcaccagttcggc 1740 gagccgctcg ctcccccgct cgtgtactac taccagaacg acgaccacgttcgcgagatg 1800 gggcatcaga agatgctcgg gcgcgacctg ctgatcgcga tcgtcgccggagagggcgag 1860 cgggaacgcg acgtgtacct tccggcgggc gagtggatcg acatccacacgaacgagcgc 1920 atccagagca cgggtcagtg gatcgacaac gtgccgctgt ggcgtgacggcgtcttcacc 1980 ctgccggcgt acgcccgggc gggggcgatc atcccgaagg ccttcgtcgacgcctccacg 2040 aaggacatca ccggcaagcg cgaggatgcc gcggtgcgca acgagctgatcgcaaccgtt 2100 tacgccgacg acgtcgcgag cgacttcacc ctgtacgagg atgacggcgcgacgaccgca 2160 tacgccgacg gggctgtcag gaccacgcag atcagccaat cgctcacgaacggcgtggcc 2220 acggtgacgg tgggagcggc atctggaacc tactccggtg cgccctccacccgtcccacg 2280 gtcgtcgagc ttgtcactga cggcacgcag gcctcgaccg tctccctcggcagcgttccg 2340 ctgacggagc acgcgaacaa ggcggcgttc gacgcggcga gcagcggctggtacaacgcc 2400 ggcggggggc tcgttgtggc caaggcggcg agcagttcgg tgaacaccgccaagaccttc 2460 tcgttcacgc tcggtgagga gtcggtctgg gcgacgttct cctgcgagaacgccacgacg 2520 accttcggtc agtcagtgta cgtcgtcgga aatgttccgc agctcggcaactggtcgccg 2580 gcggatgccg tgaagctcga gccgagcgcc taccccacct ggaccggggtggtgcggaac 2640 ctgccgccgt cgagcacggt cgaatggaag tgcatcaaac gtcaggaggccggcctgccg 2700 aacacggcgg atgcgtggga gcccggcggg aacaacatcc tctcgacgccaccttccggc 2760 tcggcgggga taaccaccgg cgccttc 2787 7 9 PRT Bacillusglobisporus C11 7 Tyr Val Ser Ser Leu Gly Asn Leu Ile 1 5 8 10 PRTBacillus globisporus C11 8 Asp Ile Tyr Gly Asp Val Thr Tyr Val Lys 1 510 9 14 PRT Bacillus globisporus C11 9 Gln Pro Trp Tyr Phe Gly Ser ThrAla Glu Glu Ala Ser Lys 1 5 10 10 31 PRT Bacillus globisporus C11 10 AlaSer Ile Asn Thr Asn His Thr Gly Tyr Thr Gly Thr Gly Phe Val 1 5 10 15Asp Gly Leu Gly Asn Asp Gly Ala Gly Val Thr Phe Tyr Pro Lys 20 25 30 1125 PRT Bacillus globisporus C11 11 Leu Leu Leu Ser Thr Ser Ala Ala GlyThr Pro Val Ser Glu Gln Asn 1 5 10 15 Leu Leu Asp Asn Pro Gly Phe GluArg 20 25 12 14 PRT Bacillus globisporus C11 12 Asp Ile Tyr Leu Pro SerGly Ser Trp Ile Asp Tyr Ala Arg 1 5 10 13 33 PRT Bacillus globisporusC11 13 Asp Ala Ser Ala Asn Val Thr Thr Gln Gly Thr Asp Ala Thr Asn Gly 15 10 15 Gly Tyr Phe Tyr Pro Gly His Asn Glu Tyr Gln Asp Tyr Phe Ile Pro20 25 30 Val 14 24 PRT Bacillus globisporus C11 14 Tyr Tyr Ser Asp AlaGly Asp Thr Gly Asn Val Asn Ile Asp Asn Ile 1 5 10 15 Thr Val Pro PheAla Pro Ile Ile 20 15 16 PRT Bacillus globisporus C11 15 Ser Thr Ser LeuAla Asn Leu Ala Asn Trp Asp Thr Trp Ser Thr Gln 1 5 10 15 16 17 PRTBacillus globisporus C11 16 Trp Ser Leu Gly Phe Met Asn Phe Glu Trp AspThr Asn Gln Thr Glu 1 5 10 15 Phe 17 20 PRT Bacillus globisporus C11 17Asn Tyr Thr Asp Ala Trp Met Phe Gly Asp Trp Leu Leu Ala Ala Pro 1 5 1015 Val Val Asp Lys 20 18 5180 DNA Bacillus globisporus C11 CDS(877)..(4728) 18 atctaccggt ttttgtgaag tttggcagta ttcttccgat gaatttgaacgcgcaatatc 60 aagtgggcgg gaccattggc aacagcttga cgagctacac gaatctcgcgttccgcattt 120 atccgcttgg gacaacaacg tacgactgga atgatgatat tggcggttcggtgaaaacca 180 taacttctac agagcaatat gggttgaata aagaaaccgt gactgttccagcgattaatt 240 ctaccaagac attgcaagtg tttacgacta agccttcctc tgtaacggtgggtggttctg 300 tgatgacaga gtacagtact ttaactgccc taacgggagc gtcgacaggctggtactatg 360 atactgtaca gaaattcact tacgtcaagc ttggttcaag tgcatctgctcaatccgttg 420 tgctaaatgg cgttaataag gtggaatatg aagcagaatt cggcgtgcaaagcggcgttt 480 caacgaacac gaaccatgca ggttatactg gtacaggatt tgtggacggctttgagactc 540 ttggagacaa tgttgctttt gatgtttccg tcaaagccgc aggtacttatacgatgaagg 600 ttcggtattc atccggtgca ggcaatggct caagagccat ctatgtgaataacaccaaag 660 tgacggacct tgccttgccg caaacaacaa gctgggatac atgggggactgctacgttta 720 gcgtctcgct gagtacaggt ctcaacacgg tgaaagtcag ctatgatggtaccagttcac 780 ttggcattaa tttcgataac atcgcgattg tagagcaata aaaggtcgggagggcaagtc 840 cctcccttaa tttctaatcg aaagggagta tccttg atg cgt cca ccaaac aaa 894 Met Arg Pro Pro Asn Lys 1 5 gaa att cca cgt att ctt gct tttttt aca gcg ttt acg ttg ttt ggt 942 Glu Ile Pro Arg Ile Leu Ala Phe PheThr Ala Phe Thr Leu Phe Gly 10 15 20 tca acc ctt gcc ttg ctt cct gct ccgcct gcg cat gcc tat gtc agc 990 Ser Thr Leu Ala Leu Leu Pro Ala Pro ProAla His Ala Tyr Val Ser 25 30 35 agc cta gga aat ctc att tct tcg agt gtcacc gga gat acc ttg acg 1038 Ser Leu Gly Asn Leu Ile Ser Ser Ser Val ThrGly Asp Thr Leu Thr 40 45 50 cta act gtt gat aac ggt gcg gag ccg agt gatgac ctc ttg att gtt 1086 Leu Thr Val Asp Asn Gly Ala Glu Pro Ser Asp AspLeu Leu Ile Val 55 60 65 70 caa gcg gtg caa aac ggt att ttg aag gtg gattat cgt cca aat agc 1134 Gln Ala Val Gln Asn Gly Ile Leu Lys Val Asp TyrArg Pro Asn Ser 75 80 85 ata acg ccg agc gcg aag acg ccg atg ctg gat ccgaac aaa act tgg 1182 Ile Thr Pro Ser Ala Lys Thr Pro Met Leu Asp Pro AsnLys Thr Trp 90 95 100 tca gct gta gga gct acg att aat acg aca gcc aatcca atg acc atc 1230 Ser Ala Val Gly Ala Thr Ile Asn Thr Thr Ala Asn ProMet Thr Ile 105 110 115 acg act tcc aat atg aag att gag att acc aag aatcca gta cga atg 1278 Thr Thr Ser Asn Met Lys Ile Glu Ile Thr Lys Asn ProVal Arg Met 120 125 130 acg gtc aag aag gcg gac ggc act acg cta ttc tgggaa cca tca ggc 1326 Thr Val Lys Lys Ala Asp Gly Thr Thr Leu Phe Trp GluPro Ser Gly 135 140 145 150 gga ggg gta ttc tca gac ggt gtg cgc ttc cttcat gcc aca ggg gat 1374 Gly Gly Val Phe Ser Asp Gly Val Arg Phe Leu HisAla Thr Gly Asp 155 160 165 aat atg tat ggc atc cgg agc ttc aat gct tttgat agc ggg ggt gac 1422 Asn Met Tyr Gly Ile Arg Ser Phe Asn Ala Phe AspSer Gly Gly Asp 170 175 180 ctg ctg cgg aat tcg tcc aat cat gcc gcc catgcg ggt gaa cag gga 1470 Leu Leu Arg Asn Ser Ser Asn His Ala Ala His AlaGly Glu Gln Gly 185 190 195 gat tcc ggt ggt ccg ctt att tgg agt acg gcagga tat gga cta tta 1518 Asp Ser Gly Gly Pro Leu Ile Trp Ser Thr Ala GlyTyr Gly Leu Leu 200 205 210 gtc gat agc gat ggc ggc tac ccc tat aca gatagc aca acc ggt caa 1566 Val Asp Ser Asp Gly Gly Tyr Pro Tyr Thr Asp SerThr Thr Gly Gln 215 220 225 230 atg gag ttt tat tat ggt ggg acc cct cctgag gga cgt cgt tat gcg 1614 Met Glu Phe Tyr Tyr Gly Gly Thr Pro Pro GluGly Arg Arg Tyr Ala 235 240 245 aaa caa aac gtg gaa tat tat att atg ctcgga acc ccc aag gaa att 1662 Lys Gln Asn Val Glu Tyr Tyr Ile Met Leu GlyThr Pro Lys Glu Ile 250 255 260 atg acc gac gta ggg gaa atc aca ggg aaaccg cct atg ctg cct aag 1710 Met Thr Asp Val Gly Glu Ile Thr Gly Lys ProPro Met Leu Pro Lys 265 270 275 tgg tcg ctt gga ttc atg aac ttt gag tgggat acg aat caa acg gag 1758 Trp Ser Leu Gly Phe Met Asn Phe Glu Trp AspThr Asn Gln Thr Glu 280 285 290 ttt acg aat aat gtg gat acg tat cgt gccaaa aat atc ccc ata gat 1806 Phe Thr Asn Asn Val Asp Thr Tyr Arg Ala LysAsn Ile Pro Ile Asp 295 300 305 310 gct tac gcc ttc gac tat gac tgg aaaaag tac ggg gaa acc aac tat 1854 Ala Tyr Ala Phe Asp Tyr Asp Trp Lys LysTyr Gly Glu Thr Asn Tyr 315 320 325 ggt gaa ttc gcg tgg aat acg act aatttc cct tct gcg tca acg act 1902 Gly Glu Phe Ala Trp Asn Thr Thr Asn PhePro Ser Ala Ser Thr Thr 330 335 340 tct tta aag tca aca atg gat gct aaaggc atc aaa atg atc gga att 1950 Ser Leu Lys Ser Thr Met Asp Ala Lys GlyIle Lys Met Ile Gly Ile 345 350 355 aca aaa ccc cgc atc gtt acg aag gatgct tca gcg aat gtg acg acc 1998 Thr Lys Pro Arg Ile Val Thr Lys Asp AlaSer Ala Asn Val Thr Thr 360 365 370 caa ggg acg gac gcg aca aat ggc ggttat ttt tat cca ggc cat aac 2046 Gln Gly Thr Asp Ala Thr Asn Gly Gly TyrPhe Tyr Pro Gly His Asn 375 380 385 390 gag tat cag gat tat ttc att cccgta act gtg cgt agt atc gat cct 2094 Glu Tyr Gln Asp Tyr Phe Ile Pro ValThr Val Arg Ser Ile Asp Pro 395 400 405 tac aat gct aac gaa cgt gct tggttc tgg aat cat tcc aca gat gcg 2142 Tyr Asn Ala Asn Glu Arg Ala Trp PheTrp Asn His Ser Thr Asp Ala 410 415 420 ctt aat aaa ggg atc gta ggt tggtgg aat gac gag acg gat aaa gta 2190 Leu Asn Lys Gly Ile Val Gly Trp TrpAsn Asp Glu Thr Asp Lys Val 425 430 435 tct tcg ggt gga gcg tta tat tggttt ggc aat ttc aca aca ggc cac 2238 Ser Ser Gly Gly Ala Leu Tyr Trp PheGly Asn Phe Thr Thr Gly His 440 445 450 atg tct cag acg atg tac gaa gggggg cgg gct tac acg agt gga gcg 2286 Met Ser Gln Thr Met Tyr Glu Gly GlyArg Ala Tyr Thr Ser Gly Ala 455 460 465 470 cag cgt gtt tgg caa acg gctaga acc ttc tac cca ggt gcc cag cgg 2334 Gln Arg Val Trp Gln Thr Ala ArgThr Phe Tyr Pro Gly Ala Gln Arg 475 480 485 tat gcg act acg ctt tgg tctggc gat att ggc att caa tac aat aaa 2382 Tyr Ala Thr Thr Leu Trp Ser GlyAsp Ile Gly Ile Gln Tyr Asn Lys 490 495 500 ggc gaa cgg atc aat tgg gctgcc ggg atg cag gag caa agg gca gtt 2430 Gly Glu Arg Ile Asn Trp Ala AlaGly Met Gln Glu Gln Arg Ala Val 505 510 515 atg cta tcc tcc gtg aac aatggc cag gtg aaa tgg ggc atg gat acc 2478 Met Leu Ser Ser Val Asn Asn GlyGln Val Lys Trp Gly Met Asp Thr 520 525 530 ggc gga ttc aat cag cag gatggc acg acg aac aat ccg aat ccc gat 2526 Gly Gly Phe Asn Gln Gln Asp GlyThr Thr Asn Asn Pro Asn Pro Asp 535 540 545 550 tta tac gct cgg tgg atgcag ttc agt gcc cta acg cct gtt ttc cga 2574 Leu Tyr Ala Arg Trp Met GlnPhe Ser Ala Leu Thr Pro Val Phe Arg 555 560 565 gtg cat ggg aac aac catcag cag cgc cag cca tgg tac ttc gga tcg 2622 Val His Gly Asn Asn His GlnGln Arg Gln Pro Trp Tyr Phe Gly Ser 570 575 580 act gcg gag gag gcc tccaaa gag gca att cag ctg cgg tac tcc ctg 2670 Thr Ala Glu Glu Ala Ser LysGlu Ala Ile Gln Leu Arg Tyr Ser Leu 585 590 595 atc cct tat atg tat gcctat gag aga agt gct tac gag aat ggg aat 2718 Ile Pro Tyr Met Tyr Ala TyrGlu Arg Ser Ala Tyr Glu Asn Gly Asn 600 605 610 ggg ctc gtt cgg cca ttgatg caa gcc tat cca aca gat gcg gcc gtc 2766 Gly Leu Val Arg Pro Leu MetGln Ala Tyr Pro Thr Asp Ala Ala Val 615 620 625 630 aaa aat tac acg gatgct tgg atg ttt ggt gac tgg ctg ctg gct gca 2814 Lys Asn Tyr Thr Asp AlaTrp Met Phe Gly Asp Trp Leu Leu Ala Ala 635 640 645 cct gtg gta gat aaacag cag acg agt aag gat atc tat tta ccg tct 2862 Pro Val Val Asp Lys GlnGln Thr Ser Lys Asp Ile Tyr Leu Pro Ser 650 655 660 ggg tca tgg att gactat gcg cga ggc aat gca ata act ggc ggt caa 2910 Gly Ser Trp Ile Asp TyrAla Arg Gly Asn Ala Ile Thr Gly Gly Gln 665 670 675 acc atc cga tat tcggtt aat ccg gac acg ttg aca gac atg cct ctc 2958 Thr Ile Arg Tyr Ser ValAsn Pro Asp Thr Leu Thr Asp Met Pro Leu 680 685 690 ttt att aaa aaa ggtgcc att att cca aca cag aaa gtg cag gat tac 3006 Phe Ile Lys Lys Gly AlaIle Ile Pro Thr Gln Lys Val Gln Asp Tyr 695 700 705 710 gta ggg cag gcttcc gtc act tcc gtt gat gtg gat gtg ttt ccg gat 3054 Val Gly Gln Ala SerVal Thr Ser Val Asp Val Asp Val Phe Pro Asp 715 720 725 acg acg cag tcgagt ttc acg tac tac gat gat gat ggc gcc agt tat 3102 Thr Thr Gln Ser SerPhe Thr Tyr Tyr Asp Asp Asp Gly Ala Ser Tyr 730 735 740 aac tat gag agcggc act tat ttt aag caa aat atg act gct cag gat 3150 Asn Tyr Glu Ser GlyThr Tyr Phe Lys Gln Asn Met Thr Ala Gln Asp 745 750 755 aat ggg tca ggctcg tta agt ttt act tta gga gca aag agt ggc agt 3198 Asn Gly Ser Gly SerLeu Ser Phe Thr Leu Gly Ala Lys Ser Gly Ser 760 765 770 tac acg ccg gctctc caa tcc tat atc gtt aag ctg cac ggt tct gct 3246 Tyr Thr Pro Ala LeuGln Ser Tyr Ile Val Lys Leu His Gly Ser Ala 775 780 785 790 gga act tctgtt acg aat aac agc gca gct atg aca tct tat gca agc 3294 Gly Thr Ser ValThr Asn Asn Ser Ala Ala Met Thr Ser Tyr Ala Ser 795 800 805 ttg gaa gcatta aaa gct gct gct ggg gaa ggc tgg gcg act ggg aag 3342 Leu Glu Ala LeuLys Ala Ala Ala Gly Glu Gly Trp Ala Thr Gly Lys 810 815 820 gac att tatggg gat gtc acc tat gtg aaa gtg acg gca ggt aca gct 3390 Asp Ile Tyr GlyAsp Val Thr Tyr Val Lys Val Thr Ala Gly Thr Ala 825 830 835 tct tct aaatct att gct gtt aca ggt gtt gct gcc gtg agc gca act 3438 Ser Ser Lys SerIle Ala Val Thr Gly Val Ala Ala Val Ser Ala Thr 840 845 850 act tcg caatac gaa gct gag gat gca tcg ctt tct ggc aat tcg gtt 3486 Thr Ser Gln TyrGlu Ala Glu Asp Ala Ser Leu Ser Gly Asn Ser Val 855 860 865 870 gct gcaaag gcg tcc ata aac acg aat cat acc gga tat acg gga act 3534 Ala Ala LysAla Ser Ile Asn Thr Asn His Thr Gly Tyr Thr Gly Thr 875 880 885 gga tttgta gat ggt ttg ggg aat gat ggc gct ggt gtc acc ttc tat 3582 Gly Phe ValAsp Gly Leu Gly Asn Asp Gly Ala Gly Val Thr Phe Tyr 890 895 900 cca aaggtg aaa act ggc ggt gac tac aat gtc tcc ttg cgt tat gcg 3630 Pro Lys ValLys Thr Gly Gly Asp Tyr Asn Val Ser Leu Arg Tyr Ala 905 910 915 aat gcttca ggc acg gct aag tca gtc agt att ttt gtt aat gga aaa 3678 Asn Ala SerGly Thr Ala Lys Ser Val Ser Ile Phe Val Asn Gly Lys 920 925 930 aga gtgaag tcc acc tcg ctc gct aat ctc gca aat tgg gac act tgg 3726 Arg Val LysSer Thr Ser Leu Ala Asn Leu Ala Asn Trp Asp Thr Trp 935 940 945 950 tctaca caa tct gag aca ctg ccg ttg acg gca ggt gtg aat gtt gtg 3774 Ser ThrGln Ser Glu Thr Leu Pro Leu Thr Ala Gly Val Asn Val Val 955 960 965 acctat aaa tat tac tcc gat gcg gga gat aca ggc aat gtt aac atc 3822 Thr TyrLys Tyr Tyr Ser Asp Ala Gly Asp Thr Gly Asn Val Asn Ile 970 975 980 gacaac atc acg gta cct ttt gcg cca att atc ggt aag tat gaa gca 3870 Asp AsnIle Thr Val Pro Phe Ala Pro Ile Ile Gly Lys Tyr Glu Ala 985 990 995 gagagt gct gag ctt tct ggt ggc agc tca ttg aac acg aac cat 3915 Glu Ser AlaGlu Leu Ser Gly Gly Ser Ser Leu Asn Thr Asn His 1000 1005 1010 tgg tactac agt ggt acg gct ttt gta gac ggt ttg agt gct gta 3960 Trp Tyr Tyr SerGly Thr Ala Phe Val Asp Gly Leu Ser Ala Val 1015 1020 1025 ggc gcg caggtg aaa tac aac gtg aat gtc cct agc gca gga agt 4005 Gly Ala Gln Val LysTyr Asn Val Asn Val Pro Ser Ala Gly Ser 1030 1035 1040 tat cag gta gcgctg cga tat gcg aat ggc agt gca gcg acg aaa 4050 Tyr Gln Val Ala Leu ArgTyr Ala Asn Gly Ser Ala Ala Thr Lys 1045 1050 1055 acg ttg agt act tatatc aat gga gcc aag ctg ggg caa acc agt 4095 Thr Leu Ser Thr Tyr Ile AsnGly Ala Lys Leu Gly Gln Thr Ser 1060 1065 1070 ttt acg agt cct ggt acgaat tgg aat gtt tgg cag gat aat gtg 4140 Phe Thr Ser Pro Gly Thr Asn TrpAsn Val Trp Gln Asp Asn Val 1075 1080 1085 caa acg gtg acg tta aat gcaggg gca aac acg att gcg ttt aaa 4185 Gln Thr Val Thr Leu Asn Ala Gly AlaAsn Thr Ile Ala Phe Lys 1090 1095 1100 tac gac gcc gct gac agc ggg aacatc aac gta gat cgt ctg ctt 4230 Tyr Asp Ala Ala Asp Ser Gly Asn Ile AsnVal Asp Arg Leu Leu 1105 1110 1115 ctt tca act tcg gca gcg gga acg ccggtt tct gag cag aac ctg 4275 Leu Ser Thr Ser Ala Ala Gly Thr Pro Val SerGlu Gln Asn Leu 1120 1125 1130 cta gac aat ccc ggt ttc gag cgt gac acgagt caa acc aat aac 4320 Leu Asp Asn Pro Gly Phe Glu Arg Asp Thr Ser GlnThr Asn Asn 1135 1140 1145 tgg att gag tgg cat cca ggc acg caa gct gttgct ttt ggc gtt 4365 Trp Ile Glu Trp His Pro Gly Thr Gln Ala Val Ala PheGly Val 1150 1155 1160 gat agc ggc tca acc acc aat ccg ccg gaa tcc ccgtgg tcg ggt 4410 Asp Ser Gly Ser Thr Thr Asn Pro Pro Glu Ser Pro Trp SerGly 1165 1170 1175 gat aag cgt gcc tac ttc ttt gca gca ggt gcc tat caacaa agc 4455 Asp Lys Arg Ala Tyr Phe Phe Ala Ala Gly Ala Tyr Gln Gln Ser1180 1185 1190 atc cat caa acc att agt gtt cct gtt aat aat gta aaa tacaaa 4500 Ile His Gln Thr Ile Ser Val Pro Val Asn Asn Val Lys Tyr Lys1195 1200 1205 ttt gaa gcc tgg gtc cgc atg aag aat acg acg ccg acg acggca 4545 Phe Glu Ala Trp Val Arg Met Lys Asn Thr Thr Pro Thr Thr Ala1210 1215 1220 aga gcc gaa att caa aac tat ggc gga tca gcc att tat gcgaac 4590 Arg Ala Glu Ile Gln Asn Tyr Gly Gly Ser Ala Ile Tyr Ala Asn1225 1230 1235 ata agt aac agc ggt gtt tgg aaa tat atc agc gta agt gatatt 4635 Ile Ser Asn Ser Gly Val Trp Lys Tyr Ile Ser Val Ser Asp Ile1240 1245 1250 atg gtg acc aat ggt cag ata gat gtt gga ttt tac gtg gattca 4680 Met Val Thr Asn Gly Gln Ile Asp Val Gly Phe Tyr Val Asp Ser1255 1260 1265 cct ggt gga act acg ctt cac att gat gat gtg cgc gta accaaa 4725 Pro Gly Gly Thr Thr Leu His Ile Asp Asp Val Arg Val Thr Lys1270 1275 1280 caa taaacaaaca accagctctc ccgttaatgg gagggctggttgtttgttat 4778 Gln gataatccat ctatttagag tggattaaac gttttgaagtgcttgctgaa cttcttgcac 4838 aatggataac gccgcggtgc gggcacttga gaaagcacgttctgcaagct ctcccttacc 4898 tgtacagccg tctccgcaga agtagaaagg aacgttttccacgcgtatcg gcagcagatt 4958 attggaagca atgtttttca cgctggaaac catcgctttcttggaaaccc gtttcacggc 5018 tgtgacatcg cgccagcctg gataatgttt atcaaataaggcttccattt ggaggttctt 5078 ctcttccagg tacgctttgc gctgctcctc gttatcaaagcggtcgctta agtatgcgat 5138 accttgcagc agctgcccgc cttctggtac tagtgtgtgatc 5180 19 8 PRT Bacillus globisporus N75 19 His Val Ser Ala Leu Gly AsnLeu 1 5 20 19 PRT Bacillus globisporus N75 20 Thr Gly Gly Asp Tyr AsnVal Ser Leu Arg Tyr Ala Asn Ser Thr Gly 1 5 10 15 Ala Ala Lys 21 20 PRTBacillus globisporus N75 21 Asp Phe Ser Asn Asn Pro Thr Val Gln Gly ThrAsp Ala Ala Ser Gly 1 5 10 15 Gly Tyr Phe Tyr 20 22 25 PRT Bacillusglobisporus N75 22 Tyr Thr Val Asn Ala Pro Ala Ala Gly Ser Tyr Gln IleAla Leu Arg 1 5 10 15 Trp Ala Asn Gly Thr Gly Ala Ala Lys 20 25 23 25PRT Bacillus globisporus N75 23 Tyr Glu Ala Glu Ser Ala Glu Leu Ser GlyGly Ser Thr Val Asn Gln 1 5 10 15 Asn His Trp Phe Tyr Ser Gly Thr Ala 2025 24 11 PRT Bacillus globisporus N75 24 Asn Tyr Leu Asp Ala Trp Met PheGly Asp Trp 1 5 10 25 4991 DNA Bacillus globisporus N75 CDS(466)..(4326) 25 ggtaccggct ttgtcgacgg cttcgatgcg gcaggcgatg cagtgaccttcgacgtatcc 60 gtcaaagcgg ccggcacgta tgcgctcaag gtccggtacg cttccgctggtggcaacgct 120 tcacgcgcta tctatgtcaa caacgccaag gtgaccgatc tggcgcttccggcaacggcc 180 aactgggaca cctgggggac ggcaaccgtc aacgtagcct taaacgccggctacaactcg 240 atcaaggtca gctacgacaa caccaatacg ctcggcatta atctcgataacattgcgatc 300 gtggagcatt gacagcagga atcttcgcga ggaatgagtt agcgaagagttcatgcaggc 360 agaggggtta cccataattg taaagcccgg cgcagccagg caccaagtatgcccgggagg 420 gccgccggcc ctccctttat ttcaatgatg aaaggcggca tcgat atg ggtcta tgg 477 Met Gly Leu Trp 1 aac aaa cga gtc act cgc atc ctc tcc gtactc gca gca agc gcg ctg 525 Asn Lys Arg Val Thr Arg Ile Leu Ser Val LeuAla Ala Ser Ala Leu 5 10 15 20 atc ggc tct acc gta cct tct cta gcg ccacct ccc gct caa gcc cat 573 Ile Gly Ser Thr Val Pro Ser Leu Ala Pro ProPro Ala Gln Ala His 25 30 35 gtg agc gcg ctg ggc aac ctg ctt tcc tcg gcggtg acc ggg gat acg 621 Val Ser Ala Leu Gly Asn Leu Leu Ser Ser Ala ValThr Gly Asp Thr 40 45 50 ctc acg ctg acg atc gat aac ggc gcg gaa ccg aatgac gat att cta 669 Leu Thr Leu Thr Ile Asp Asn Gly Ala Glu Pro Asn AspAsp Ile Leu 55 60 65 gtt ctg caa gca gtc cag aac ggt att ctg aag gtg gactac cgg ccg 717 Val Leu Gln Ala Val Gln Asn Gly Ile Leu Lys Val Asp TyrArg Pro 70 75 80 aac ggt gta gct cca agc gcg gat acg ccg atg ctg gat cccaat aaa 765 Asn Gly Val Ala Pro Ser Ala Asp Thr Pro Met Leu Asp Pro AsnLys 85 90 95 100 acc tgg ccg tcc ata ggc gcc gtt atc aat aca gcc tct aatccg atg 813 Thr Trp Pro Ser Ile Gly Ala Val Ile Asn Thr Ala Ser Asn ProMet 105 110 115 acg atc aca acg ccg gcg atg aag att gag att gcc aaa aatccg gtg 861 Thr Ile Thr Thr Pro Ala Met Lys Ile Glu Ile Ala Lys Asn ProVal 120 125 130 cgc ctg acc gtg aaa aaa ccg gac ggc acc gct ctg tta tgggaa ccc 909 Arg Leu Thr Val Lys Lys Pro Asp Gly Thr Ala Leu Leu Trp GluPro 135 140 145 ccg acc ggc ggc gtc ttc tcg gac ggc gtc cgt ttc ttg cacggg acg 957 Pro Thr Gly Gly Val Phe Ser Asp Gly Val Arg Phe Leu His GlyThr 150 155 160 ggc gac aat atg tac ggc atc cgc agc ttc aat gct ttt gacagc ggc 1005 Gly Asp Asn Met Tyr Gly Ile Arg Ser Phe Asn Ala Phe Asp SerGly 165 170 175 180 ggg gat ctg ctg cgc aac agc tcc acc caa gcc gcc cgtgca ggc gac 1053 Gly Asp Leu Leu Arg Asn Ser Ser Thr Gln Ala Ala Arg AlaGly Asp 185 190 195 cag ggc aac tcc ggc ggc ccg ctg atc tgg agc aca gccggg tac ggg 1101 Gln Gly Asn Ser Gly Gly Pro Leu Ile Trp Ser Thr Ala GlyTyr Gly 200 205 210 gtg ctc gtt gac agc gac ggt ggg tat ccg ttc acg gacgag gct acc 1149 Val Leu Val Asp Ser Asp Gly Gly Tyr Pro Phe Thr Asp GluAla Thr 215 220 225 ggc aag ctg gag ttc tat tac ggc ggc acg cct ccg gaaggc cgg cgc 1197 Gly Lys Leu Glu Phe Tyr Tyr Gly Gly Thr Pro Pro Glu GlyArg Arg 230 235 240 tat acg aag cag gat gtg gag tac tac atc atg ctc ggcacg ccg aaa 1245 Tyr Thr Lys Gln Asp Val Glu Tyr Tyr Ile Met Leu Gly ThrPro Lys 245 250 255 260 gag atc atg tcc ggc gtc ggg gaa att acg ggc aaaccg ccg atg ctg 1293 Glu Ile Met Ser Gly Val Gly Glu Ile Thr Gly Lys ProPro Met Leu 265 270 275 ccc aag tgg tcc ctg ggc ttt atg aac ttc gag tgggat ctg aat gaa 1341 Pro Lys Trp Ser Leu Gly Phe Met Asn Phe Glu Trp AspLeu Asn Glu 280 285 290 gct gag ctc aag aac cat gtg gat acg tac cgg gccaaa aat att ccg 1389 Ala Glu Leu Lys Asn His Val Asp Thr Tyr Arg Ala LysAsn Ile Pro 295 300 305 atc gac ggc tat gcg atc gat ttc gat tgg aag aagtac ggc gag aat 1437 Ile Asp Gly Tyr Ala Ile Asp Phe Asp Trp Lys Lys TyrGly Glu Asn 310 315 320 aat tac ggc gaa ttc gct tgg aat acg gcc aat ttccct tcc gcc gcc 1485 Asn Tyr Gly Glu Phe Ala Trp Asn Thr Ala Asn Phe ProSer Ala Ala 325 330 335 340 acg acg gcg ctg aag tcg cag atg gac gcc aagggc att aaa atg atc 1533 Thr Thr Ala Leu Lys Ser Gln Met Asp Ala Lys GlyIle Lys Met Ile 345 350 355 ggc ata acc aag cct cgc atc gcg acg aag gatttt tcg aac aat cct 1581 Gly Ile Thr Lys Pro Arg Ile Ala Thr Lys Asp PheSer Asn Asn Pro 360 365 370 acc gtg cag gga acg gac gcg gcg agc ggc ggttat ttt tat ccg gga 1629 Thr Val Gln Gly Thr Asp Ala Ala Ser Gly Gly TyrPhe Tyr Pro Gly 375 380 385 cat agc gaa tac aag gac tac ttc atc ccg gtcttt gtg cgc agc atc 1677 His Ser Glu Tyr Lys Asp Tyr Phe Ile Pro Val PheVal Arg Ser Ile 390 395 400 gac cct tat aac cct gct gca cgc tcc tgg ttctgg aac cac tcc aag 1725 Asp Pro Tyr Asn Pro Ala Ala Arg Ser Trp Phe TrpAsn His Ser Lys 405 410 415 420 gat gcg ttc gat aaa ggc atc gta ggc tggtgg aac gac gag acg gat 1773 Asp Ala Phe Asp Lys Gly Ile Val Gly Trp TrpAsn Asp Glu Thr Asp 425 430 435 gcg gta tcg tcg gga ggg gcc tcc tac tggttc ggc aat ttt acg acc 1821 Ala Val Ser Ser Gly Gly Ala Ser Tyr Trp PheGly Asn Phe Thr Thr 440 445 450 ggc cat atg tcc cag gcg ctt tac gag ggacag cgg gca tat acg tcg 1869 Gly His Met Ser Gln Ala Leu Tyr Glu Gly GlnArg Ala Tyr Thr Ser 455 460 465 aac gcc cag cgc gtc tgg cag aca gcg cgcacg ttc tat ccc ggg gcg 1917 Asn Ala Gln Arg Val Trp Gln Thr Ala Arg ThrPhe Tyr Pro Gly Ala 470 475 480 cag cgt tat gcg acg acg ctc tgg tcg ggagac atc ggg att cag tat 1965 Gln Arg Tyr Ala Thr Thr Leu Trp Ser Gly AspIle Gly Ile Gln Tyr 485 490 495 500 acc aag ggg gaa aga atc aac tgg gctgcc ggc atg cag gag cag cgg 2013 Thr Lys Gly Glu Arg Ile Asn Trp Ala AlaGly Met Gln Glu Gln Arg 505 510 515 gcg gtg atg ctt tct tcg atc aac aacggc cag gtc aaa tgg gga atg 2061 Ala Val Met Leu Ser Ser Ile Asn Asn GlyGln Val Lys Trp Gly Met 520 525 530 gac aca ggc ggc ttc aac cag cag gacggc acg acg aac aat ccg aat 2109 Asp Thr Gly Gly Phe Asn Gln Gln Asp GlyThr Thr Asn Asn Pro Asn 535 540 545 ccg gac ctg tac gcc aga tgg atg cagttc agc gcg ctg act ccg gtg 2157 Pro Asp Leu Tyr Ala Arg Trp Met Gln PheSer Ala Leu Thr Pro Val 550 555 560 ttc cgc gtg cat ggc aac aat cac cagcag cgc cag cct tgg tat tat 2205 Phe Arg Val His Gly Asn Asn His Gln GlnArg Gln Pro Trp Tyr Tyr 565 570 575 580 ggc tcg aca gcc gag gag gca tccaag gaa gcg ctc cag ctc cgt tac 2253 Gly Ser Thr Ala Glu Glu Ala Ser LysGlu Ala Leu Gln Leu Arg Tyr 585 590 595 tcc ctg att cct tat atg tat gcttac gaa aga agc gcc tac gag aac 2301 Ser Leu Ile Pro Tyr Met Tyr Ala TyrGlu Arg Ser Ala Tyr Glu Asn 600 605 610 ggt aac gga ctt gtc cgg ccg ctgatg cag gaa tac cct gcc gat gcc 2349 Gly Asn Gly Leu Val Arg Pro Leu MetGln Glu Tyr Pro Ala Asp Ala 615 620 625 aac gcc aaa aac tat ctc gat gcctgg atg ttc ggc gat tgg ctg ctg 2397 Asn Ala Lys Asn Tyr Leu Asp Ala TrpMet Phe Gly Asp Trp Leu Leu 630 635 640 gcg gcg cct gtg gtc gag aag cagcag acc tcc aag gaa atc tat ctc 2445 Ala Ala Pro Val Val Glu Lys Gln GlnThr Ser Lys Glu Ile Tyr Leu 645 650 655 660 cct gca ggc act tgg att gactac aac cgg ggc acg gtg ctc acc ggc 2493 Pro Ala Gly Thr Trp Ile Asp TyrAsn Arg Gly Thr Val Leu Thr Gly 665 670 675 ggc cag aag atc agc tac gccgtc aat ccc gac acg ctg acg gat att 2541 Gly Gln Lys Ile Ser Tyr Ala ValAsn Pro Asp Thr Leu Thr Asp Ile 680 685 690 ccg ctc ttc att aag aag ggcgcg att atc cct tcg cag aag gtg cag 2589 Pro Leu Phe Ile Lys Lys Gly AlaIle Ile Pro Ser Gln Lys Val Gln 695 700 705 gac tac gtg ggc cag gct cccgtc caa acg gtg gat gtg gat gta ttc 2637 Asp Tyr Val Gly Gln Ala Pro ValGln Thr Val Asp Val Asp Val Phe 710 715 720 ccg aat acg gca caa tcg agcttt acc tat tat gac gat gac ggc agc 2685 Pro Asn Thr Ala Gln Ser Ser PheThr Tyr Tyr Asp Asp Asp Gly Ser 725 730 735 740 agc tac aat tat gaa agcgga gct tac ttc aag caa ttg atg acg gct 2733 Ser Tyr Asn Tyr Glu Ser GlyAla Tyr Phe Lys Gln Leu Met Thr Ala 745 750 755 cag gac aac gga tcc ggtgcg ctg agc ttt acg ctg ggc gcc aaa acc 2781 Gln Asp Asn Gly Ser Gly AlaLeu Ser Phe Thr Leu Gly Ala Lys Thr 760 765 770 ggc acg tac agc ccc gcactg caa tcc tat atc gtc aag ctt cac ggg 2829 Gly Thr Tyr Ser Pro Ala LeuGln Ser Tyr Ile Val Lys Leu His Gly 775 780 785 gcc gca ggc gcg tcg gtgaca agc aat ggg gcg gcg ctg gcc tcc tat 2877 Ala Ala Gly Ala Ser Val ThrSer Asn Gly Ala Ala Leu Ala Ser Tyr 790 795 800 gcc agc ctg caa gcg ctgaaa gcc tca gcc agt gaa ggc tgg gcc aag 2925 Ala Ser Leu Gln Ala Leu LysAla Ser Ala Ser Glu Gly Trp Ala Lys 805 810 815 820 ggc aag gac atc tacggc gat gtc acg tat gtc aag cta tcc gcg ggg 2973 Gly Lys Asp Ile Tyr GlyAsp Val Thr Tyr Val Lys Leu Ser Ala Gly 825 830 835 gca gcg gcg gcc aaggcg att gcc gtc acc ggc aac agc ccg gtc agc 3021 Ala Ala Ala Ala Lys AlaIle Ala Val Thr Gly Asn Ser Pro Val Ser 840 845 850 gtg gcg gat gtg cagtac gaa gcc gaa gaa gct tcg ctg tcc ggc aat 3069 Val Ala Asp Val Gln TyrGlu Ala Glu Glu Ala Ser Leu Ser Gly Asn 855 860 865 acg aca gca acc aaggcg acc gtg aat acg aac cac gca ggc tac acg 3117 Thr Thr Ala Thr Lys AlaThr Val Asn Thr Asn His Ala Gly Tyr Thr 870 875 880 ggc agc ggc ttc gtggat gga ctg agt aat ccg gga gcg gcg gtt acg 3165 Gly Ser Gly Phe Val AspGly Leu Ser Asn Pro Gly Ala Ala Val Thr 885 890 895 900 ttc tat ccg aaggtg aaa acg ggc gga gac tac aat gtc tcg ctg cgc 3213 Phe Tyr Pro Lys ValLys Thr Gly Gly Asp Tyr Asn Val Ser Leu Arg 905 910 915 tac gct aat tcgacg gga gcg gca aag agc gtc agc atc ttc gtt aac 3261 Tyr Ala Asn Ser ThrGly Ala Ala Lys Ser Val Ser Ile Phe Val Asn 920 925 930 ggc aag cgc gtcaaa tcc acg tcg ctg gcg aac ctg ccg aac tgg gat 3309 Gly Lys Arg Val LysSer Thr Ser Leu Ala Asn Leu Pro Asn Trp Asp 935 940 945 acg tgg ggg acgcag gct gag aca ctg ccg ctg acg gcg ggg acg aac 3357 Thr Trp Gly Thr GlnAla Glu Thr Leu Pro Leu Thr Ala Gly Thr Asn 950 955 960 gtt gtc acc tacaag ttc tac tcg gat gcc gga gat acg ggc tcg gtt 3405 Val Val Thr Tyr LysPhe Tyr Ser Asp Ala Gly Asp Thr Gly Ser Val 965 970 975 980 aac ctg gacaac atc acg gtg ccc ttc gct ccg gcc atc ggc aaa tac 3453 Asn Leu Asp AsnIle Thr Val Pro Phe Ala Pro Ala Ile Gly Lys Tyr 985 990 995 gag gcg gagagc gcc gag ctg agc ggc ggc agc acg gtc aac cag 3498 Glu Ala Glu Ser AlaGlu Leu Ser Gly Gly Ser Thr Val Asn Gln 1000 1005 1010 aat cat tgg ttctac agc ggc acg gca ttt gta gat ggc tta acc 3543 Asn His Trp Phe Tyr SerGly Thr Ala Phe Val Asp Gly Leu Thr 1015 1020 1025 gca ccg ggc gcc caagtc aaa tat acc gtg aac gcc ccg gcc gca 3588 Ala Pro Gly Ala Gln Val LysTyr Thr Val Asn Ala Pro Ala Ala 1030 1035 1040 ggc agc tac cag atc gcgctt cgc tat gcg aac ggc acg ggt gct 3633 Gly Ser Tyr Gln Ile Ala Leu ArgTyr Ala Asn Gly Thr Gly Ala 1045 1050 1055 gcg aag acg ctc agc acg tatgtg aac ggg acg aag ctg ggg caa 3678 Ala Lys Thr Leu Ser Thr Tyr Val AsnGly Thr Lys Leu Gly Gln 1060 1065 1070 acg gcc ttc gcc agc cct ggc ggcaac tgg aac gtg tgg cag gac 3723 Thr Ala Phe Ala Ser Pro Gly Gly Asn TrpAsn Val Trp Gln Asp 1075 1080 1085 agc gtg cag acc gtc gcg ctc gcc gccggt acg aac acg atc gcg 3768 Ser Val Gln Thr Val Ala Leu Ala Ala Gly ThrAsn Thr Ile Ala 1090 1095 1100 ttc aag tac gat gcc ggc gac agc ggc agcggc agc gtc aat ctg 3813 Phe Lys Tyr Asp Ala Gly Asp Ser Gly Ser Gly SerVal Asn Leu 1105 1110 1115 gac cgt ctg ttg ctc tct gcc gca gcg cca ggcgtg ccc gtg tcc 3858 Asp Arg Leu Leu Leu Ser Ala Ala Ala Pro Gly Val ProVal Ser 1120 1125 1130 gag cag aac ctg ctc gat aac ggg ggc ttt gaa cgcgat ccg tcg 3903 Glu Gln Asn Leu Leu Asp Asn Gly Gly Phe Glu Arg Asp ProSer 1135 1140 1145 cag agc agc aac tgg acc gag tgg cat ccg gct tcg caggcg att 3948 Gln Ser Ser Asn Trp Thr Glu Trp His Pro Ala Ser Gln Ala Ile1150 1155 1160 gct tac ggc atc gac agc ggc tcc ggg atg aat ccg cct gaatcg 3993 Ala Tyr Gly Ile Asp Ser Gly Ser Gly Met Asn Pro Pro Glu Ser1165 1170 1175 cca tgg gca ggc gat aag cgc gcc tat ttc tat gcg gca ggcccg 4038 Pro Trp Ala Gly Asp Lys Arg Ala Tyr Phe Tyr Ala Ala Gly Pro1180 1185 1190 tat cag caa agc atc cat caa aca gtc agc gtg cct gtc aataat 4083 Tyr Gln Gln Ser Ile His Gln Thr Val Ser Val Pro Val Asn Asn1195 1200 1205 gcc aag tac aag ttc gaa gcc tgg gta ttg ctg aag aat acaaca 4128 Ala Lys Tyr Lys Phe Glu Ala Trp Val Leu Leu Lys Asn Thr Thr1210 1215 1220 ccg aca acg gcc cgg gtg gag att caa aat tac ggc ggt tcgccg 4173 Pro Thr Thr Ala Arg Val Glu Ile Gln Asn Tyr Gly Gly Ser Pro1225 1230 1235 atc ttc acg aac atc agt aaa gac ggc gtc tgg aaa tac atcagc 4218 Ile Phe Thr Asn Ile Ser Lys Asp Gly Val Trp Lys Tyr Ile Ser1240 1245 1250 gtc agc gat att cag gtc acg aac ggc caa atc gat att ggcttc 4263 Val Ser Asp Ile Gln Val Thr Asn Gly Gln Ile Asp Ile Gly Phe1255 1260 1265 tat gtg gat tcg ccc gga ggc acc acg ctc cac atc gac gatgtg 4308 Tyr Val Asp Ser Pro Gly Gly Thr Thr Leu His Ile Asp Asp Val1270 1275 1280 cgg gtc acc aag caa taa tccggtaaca ctagccctcc cccgccttgc4356 Arg Val Thr Lys Gln 1285 ggcaggaggg ctttttgctt ctgtaggttgtgaaggcgat accgagcgat gagaattcga 4416 ttctgaacag ctcgccctgt gtcctgctaaattcctctcc tccctggcag ggaagccgct 4476 tccacatgtc gaattgggga ggtactatgagaagttagta ctaccgtctg caacggcttt 4536 cgctacaatg gaaccaataa gacatcgcgaaggtttggga ggattcggca tgcagagacg 4596 cgaggttaaa gtaataggca cgggcaaatatttgcccgcc catcgagtga ctgcgcagga 4656 gatggaccgg cggctaggag tgcccgacggatgggtgctg aagaagtcgg atgtggccgt 4716 tcgttatttc gccggtacgg agaaggcctcggagatgggg gcgagagcgg ctgaggcggc 4776 gctggcttcc gcaggcctgg ccttcacggatatcgactgc ctgatgtgcg ccagcgggac 4836 gatggaacag ccgattccat gcacggcggcgctcattcag aaggcgatag gccaaggaca 4896 ctccggagtg ccggcactgg atttgaatacaacctgtctg agctttgtgg cggctctgga 4956 catggtttct tatatggtga cggcgggaaggtacc 4991 26 13 PRT Arthrobacter globiformis A19 26 Ala Pro Leu Gly ValGln Arg Ala Gln Phe Gln Ser Gly 1 5 10 27 13 PRT Arthrobacterglobiformis A19 27 Gln Glu Trp Asn Leu Thr Gly Asp Pro Trp Thr Val Arg 15 10 28 30 PRT Arthrobacter globiformis A19 28 Ile Ala Asp Leu Ala GluAsp Gly Val Gly Ile Ile Pro Ile Glu Glu 1 5 10 15 Ser Tyr Val Gly ArgAsn Leu Pro Glu His Ala Arg Met Ala 20 25 30 29 30 PRT Arthrobacterglobiformis A19 29 Gly Gly Met Ile Asp Trp Thr Gln Pro Glu Ala Gly AlaVal Trp His 1 5 10 15 Asp Glu Gln Arg Gln His Leu Val Asp Glu Gly ValLeu Gly 20 25 30 30 27 PRT Arthrobacter globiformis A19 30 His Asp TyrAla Gly Pro Asp Val Phe Thr Gln Thr Gly Ser Val Leu 1 5 10 15 Gln ThrAla Ala Met Arg Ile Glu Val Asp Pro 20 25 31 27 PRT Arthrobacterglobiformis A19 31 Met Leu Gly Arg Asp Leu Leu Ile Ala Ile Val Ala GlyGlu Gly Glu 1 5 10 15 Arg Glu Arg Asp Val Tyr Leu Pro Ala Gly Glu 20 2532 5811 DNA Arthrobacter globiformis A19 CDS (1655)..(4552) 32ggtacctcgt cgaggagctc ggtgtcgacg gcttcaagac cgacgggagc gaggcgctct 60tcgggcgtga cctgatcgtc agcgacgggc gccgcggtga cgagatgcac aacgcctacc 120cgaacgagta cacctccgcc tacaacgact tcgtgcagga gacgacgggc gccgacggca 180cgatcttcag ccgggcgggc acctccggcg gccagagcga atccatcttc tgggccgggg 240accaggcgtc gacgttcggc gctttccagg aggccgtccg ggccgggcag agcgcgggcc 300agtcgggagt gccgttctgg gcctgggacc tcggcggctt caccgggtcg ttcccaagcg 360cggagctgta tctgcgctcg accgctcagg cggtgttctc gccgatcatg cagtaccact 420cggagaaggc cgaccccagt ccgtccgagg cgcgcacgcc ctggaacgtg caggcgcgca 480ccgggaacac cactgtcgtc cccaccttcg cccgttacgc gaacgtacgg atgaacctcg 540tgccctatct gtacacggag gcggacgaca gcgcgacgac gggtgtgccg atgatgcgcg 600cgatgagcct cgcgttcccc gacgacccgg atgccgcgca gtacgaccag cagtacatgt 660tcgggtctca gctgctggtc gcaccgatta cgaaccaggg ccagaccgtg aaagacgtct 720acctgcccgc gggcgagtgg tacgacttct ggaacggcgg acgcgcgagc ggcgagggcg 780tgaagatgta cgacgccgga cccgacggca tccccgtata cgctcgcgcc ggagcggtca 840tcccgctcaa cctcaacgac gcgtatgagg tgggcggcac gatcggcaac gacgtggaga 900gctacgacaa ccttgtgttc cgcgtttacc cctccggtga gagcagctac gagtacttcg 960aagaccaagc gaacgcgcac cgccggatcg atgtctcggc cgaccgcgca gcgcgcacgg 1020tcgaggtgtc tgctcccgcg ctcacgaccg cgagcacctt ccaggtgtcg ggcaccaagc 1080ccgacaccgt gaccgtcgcg ggctcggcac tgcctgaggt caacagcgtg agcgcgctgg 1140ccgcatccac cgaggcctgg tactgggatg cgaagcagca gctgacgtac gtgaaggtcg 1200gtgcgagcac cggcgagcgc acgatcctcc tgctgggcgt cgacaaggcc gggtacgagg 1260ccgagttcgc gggtcatacg gccgtctcga cgaacgccga ccacccgggc tacaccgggc 1320tcggcttcgt cgacggcttc gcgaacgcag gagacgcggt ggagttcgac gtgtgggccg 1380aggagaacgg cgcgcaccag ctccgcttcc gctacggaaa cggagcggcg acccccgcca 1440cccgcacgat ccgggtcgac ggagcgcctc tgggaacgct gtcgcttccg cccaccgggt 1500cgtggagttc gtggggcacg gcctcgatcg acgtgaccct cccacccgga cgccacgccg 1560tacggatcga gtacgccgga ggcgattccg gcggcgtcaa cctcgacaac ctcgtcctcg 1620cgcgctgagc gcacacggga aagggagaag aacc atg cct gct ctt ccg tgg cgc 1675Met Pro Ala Leu Pro Trp Arg 1 5 cgc acg acg gcg ctc gcg ctc acc acg gcggtg acg gcc gcg acc ctg 1723 Arg Thr Thr Ala Leu Ala Leu Thr Thr Ala ValThr Ala Ala Thr Leu 10 15 20 gtc gcc gtc ggg gtg aac gac gcc ggt cag gcggcg gct gct ccc ctg 1771 Val Ala Val Gly Val Asn Asp Ala Gly Gln Ala AlaAla Ala Pro Leu 25 30 35 ggc gtg caa cgc gcg cag ttc cag tcg ggg tcg agctac ctc gtc gtc 1819 Gly Val Gln Arg Ala Gln Phe Gln Ser Gly Ser Ser TyrLeu Val Val 40 45 50 55 gag gtg ctc gat gac gac ctc gtc cac ttc gag ctggcc ggg ggc ggc 1867 Glu Val Leu Asp Asp Asp Leu Val His Phe Glu Leu AlaGly Gly Gly 60 65 70 acc gcc ccc ggc acg ggc tcc ccg ctg ttc acg acg cctcag gtc gcg 1915 Thr Ala Pro Gly Thr Gly Ser Pro Leu Phe Thr Thr Pro GlnVal Ala 75 80 85 aag cac gac tac gcg gga ccc gac gtg ttc acc cag acc gggtct gtt 1963 Lys His Asp Tyr Ala Gly Pro Asp Val Phe Thr Gln Thr Gly SerVal 90 95 100 ctg cag acc gcg gcg atg cgc atc gag gtc gat ccc gcg gatctg tgc 2011 Leu Gln Thr Ala Ala Met Arg Ile Glu Val Asp Pro Ala Asp LeuCys 105 110 115 gtg acg gcc acc gac atc acc cgc acc ccg aac ctt gta ctgcac gag 2059 Val Thr Ala Thr Asp Ile Thr Arg Thr Pro Asn Leu Val Leu HisGlu 120 125 130 135 gcg tgt ccc gcc gac ctc ggc cag gcg tgg aag ggg ctgaac atc acg 2107 Ala Cys Pro Ala Asp Leu Gly Gln Ala Trp Lys Gly Leu AsnIle Thr 140 145 150 agg tcg gcg atg gag aac gcc tac ggt ctc ggg cag cagttc ttc acg 2155 Arg Ser Ala Met Glu Asn Ala Tyr Gly Leu Gly Gln Gln PhePhe Thr 155 160 165 ggc ggc agc gcg gac ggc gac tgg gtg ggc cgc acc cgcacc ccg ggt 2203 Gly Gly Ser Ala Asp Gly Asp Trp Val Gly Arg Thr Arg ThrPro Gly 170 175 180 ggc acc tac ggc aac gcg atg gtg ttc gac ccc gag aacggg ccg gtc 2251 Gly Thr Tyr Gly Asn Ala Met Val Phe Asp Pro Glu Asn GlyPro Val 185 190 195 ggc aac acg cag atc ccg gtg ctc ttc gcg gtc ggc gatgac aac gcg 2299 Gly Asn Thr Gln Ile Pro Val Leu Phe Ala Val Gly Asp AspAsn Ala 200 205 210 215 aac tac ggg ctg ttc gtc gat cag ctg tac aag caggaa tgg aac ctc 2347 Asn Tyr Gly Leu Phe Val Asp Gln Leu Tyr Lys Gln GluTrp Asn Leu 220 225 230 acc ggc gac ccg tgg acg gtg cgc atg tgg ggc gaccag gtg cgc tgg 2395 Thr Gly Asp Pro Trp Thr Val Arg Met Trp Gly Asp GlnVal Arg Trp 235 240 245 tac ctc atg agc ggc gac gac ctg ccc gac ctt cgccac gac tac atg 2443 Tyr Leu Met Ser Gly Asp Asp Leu Pro Asp Leu Arg HisAsp Tyr Met 250 255 260 gag ctg acg ggc acc ccg ccc gtg ccg ccg aag aaggcg ttc ggg ctc 2491 Glu Leu Thr Gly Thr Pro Pro Val Pro Pro Lys Lys AlaPhe Gly Leu 265 270 275 tgg gtg tcg gag ttc ggc tac gac aac tgg agc gaggtc gac aat acg 2539 Trp Val Ser Glu Phe Gly Tyr Asp Asn Trp Ser Glu ValAsp Asn Thr 280 285 290 295 atc gcg ggc ctg cgc tcg gcc gac ttt ccg gtcgat ggc gcg atg ctc 2587 Ile Ala Gly Leu Arg Ser Ala Asp Phe Pro Val AspGly Ala Met Leu 300 305 310 gac gta cag tgg ttc ggg ggc gtc acc gcc gactcg gac gac acc cgc 2635 Asp Val Gln Trp Phe Gly Gly Val Thr Ala Asp SerAsp Asp Thr Arg 315 320 325 atg ggc acc ctc gat tgg gac acg tcg agg tttccc gac cct gcg gga 2683 Met Gly Thr Leu Asp Trp Asp Thr Ser Arg Phe ProAsp Pro Ala Gly 330 335 340 aag atc gcc gac ctc gcc gag gac ggc gtc ggcatc atc ccg atc gag 2731 Lys Ile Ala Asp Leu Ala Glu Asp Gly Val Gly IleIle Pro Ile Glu 345 350 355 gag tcg tac gtc ggt cgc aac ctg ccg gag cacgcc cgg atg gcg gcg 2779 Glu Ser Tyr Val Gly Arg Asn Leu Pro Glu His AlaArg Met Ala Ala 360 365 370 375 gac ggt tac ctc gtg cgc tcc ggc tgc gctacg tgc ccg ccg gtg tac 2827 Asp Gly Tyr Leu Val Arg Ser Gly Cys Ala ThrCys Pro Pro Val Tyr 380 385 390 ctg acg ggg aac ccc tgg tgg ggc aag ggcggg atg atc gac tgg acg 2875 Leu Thr Gly Asn Pro Trp Trp Gly Lys Gly GlyMet Ile Asp Trp Thr 395 400 405 cag ccg gaa gcc ggc gcc gtc tgg cac gacgag cag cgc cag cat ctc 2923 Gln Pro Glu Ala Gly Ala Val Trp His Asp GluGln Arg Gln His Leu 410 415 420 gtc gac gag ggc gta ctg ggc cac tgg ctcgat ctc ggc gaa ccg gag 2971 Val Asp Glu Gly Val Leu Gly His Trp Leu AspLeu Gly Glu Pro Glu 425 430 435 atg tac gac ccg aac gac tgg acc gcc ggcgtc atc ccc ggc aag cac 3019 Met Tyr Asp Pro Asn Asp Trp Thr Ala Gly ValIle Pro Gly Lys His 440 445 450 455 gcg cac gcc gac tat cac aac gcg tacaac ctg ctg tgg gcg cag agc 3067 Ala His Ala Asp Tyr His Asn Ala Tyr AsnLeu Leu Trp Ala Gln Ser 460 465 470 atc gcc gac ggg tac gcc gac aac ggcgtg cag aag cgt ccc ttc atg 3115 Ile Ala Asp Gly Tyr Ala Asp Asn Gly ValGln Lys Arg Pro Phe Met 475 480 485 ctg acg cgc gcc gcg gcc gcc ggc atccag cgt cat ggc gcg ggc atg 3163 Leu Thr Arg Ala Ala Ala Ala Gly Ile GlnArg His Gly Ala Gly Met 490 495 500 tgg tca gcc gac atc ggg tcg acc atgaag gcg ctc ggg agc cag cag 3211 Trp Ser Ala Asp Ile Gly Ser Thr Met LysAla Leu Gly Ser Gln Gln 505 510 515 aac gcg cag atg cac atg tcg atg tcgggg atc gac tat tac ggc tcc 3259 Asn Ala Gln Met His Met Ser Met Ser GlyIle Asp Tyr Tyr Gly Ser 520 525 530 535 gac atc ggc ggg ttc cgg cgg gagatg gcc gac ggc gac gtg aac gag 3307 Asp Ile Gly Gly Phe Arg Arg Glu MetAla Asp Gly Asp Val Asn Glu 540 545 550 ctc tac acc cag tgg ttc gcc gacagc gcg tgg ttc gac act ccg ctc 3355 Leu Tyr Thr Gln Trp Phe Ala Asp SerAla Trp Phe Asp Thr Pro Leu 555 560 565 cgg ccg cac acc gac aat ctc tgcaac tgc ctc gag acg agc ccc gac 3403 Arg Pro His Thr Asp Asn Leu Cys AsnCys Leu Glu Thr Ser Pro Asp 570 575 580 tcg atc ggc gac gtc gcg agc aaccgc gag aac ctg gtg cgc cgc tac 3451 Ser Ile Gly Asp Val Ala Ser Asn ArgGlu Asn Leu Val Arg Arg Tyr 585 590 595 gag ctg gct ccg tac tac tac tcgctc gcg cac cgc gct cac cag ttc 3499 Glu Leu Ala Pro Tyr Tyr Tyr Ser LeuAla His Arg Ala His Gln Phe 600 605 610 615 ggc gag ccg ctc gct ccc ccgctc gtg tac tac tac cag aac gac gac 3547 Gly Glu Pro Leu Ala Pro Pro LeuVal Tyr Tyr Tyr Gln Asn Asp Asp 620 625 630 cac gtt cgc gag atg ggg catcag aag atg ctc ggg cgc gac ctg ctg 3595 His Val Arg Glu Met Gly His GlnLys Met Leu Gly Arg Asp Leu Leu 635 640 645 atc gcg atc gtc gcc gga gagggc gag cgg gaa cgc gac gtg tac ctt 3643 Ile Ala Ile Val Ala Gly Glu GlyGlu Arg Glu Arg Asp Val Tyr Leu 650 655 660 ccg gcg ggc gag tgg atc gacatc cac acg aac gag cgc atc cag agc 3691 Pro Ala Gly Glu Trp Ile Asp IleHis Thr Asn Glu Arg Ile Gln Ser 665 670 675 acg ggt cag tgg atc gac aacgtg ccg ctg tgg cgt gac ggc gtc ttc 3739 Thr Gly Gln Trp Ile Asp Asn ValPro Leu Trp Arg Asp Gly Val Phe 680 685 690 695 acc ctg ccg gcg tac gcccgg gcg ggg gcg atc atc ccg aag gcc ttc 3787 Thr Leu Pro Ala Tyr Ala ArgAla Gly Ala Ile Ile Pro Lys Ala Phe 700 705 710 gtc gac gcc tcc acg aaggac atc acc ggc aag cgc gag gat gcc gcg 3835 Val Asp Ala Ser Thr Lys AspIle Thr Gly Lys Arg Glu Asp Ala Ala 715 720 725 gtg cgc aac gag ctg atcgca acc gtt tac gcc gac gac gtc gcg agc 3883 Val Arg Asn Glu Leu Ile AlaThr Val Tyr Ala Asp Asp Val Ala Ser 730 735 740 gac ttc acc ctg tac gaggat gac ggc gcg acg acc gca tac gcc gac 3931 Asp Phe Thr Leu Tyr Glu AspAsp Gly Ala Thr Thr Ala Tyr Ala Asp 745 750 755 ggg gct gtc agg acc acgcag atc agc caa tcg ctc acg aac ggc gtg 3979 Gly Ala Val Arg Thr Thr GlnIle Ser Gln Ser Leu Thr Asn Gly Val 760 765 770 775 gcc acg gtg acg gtggga gcg gca tct gga acc tac tcc ggt gcg ccc 4027 Ala Thr Val Thr Val GlyAla Ala Ser Gly Thr Tyr Ser Gly Ala Pro 780 785 790 tcc acc cgt ccc acggtc gtc gag ctt gtc act gac ggc acg cag gcc 4075 Ser Thr Arg Pro Thr ValVal Glu Leu Val Thr Asp Gly Thr Gln Ala 795 800 805 tcg acc gtc tcc ctcggc agc gtt ccg ctg acg gag cac gcg aac aag 4123 Ser Thr Val Ser Leu GlySer Val Pro Leu Thr Glu His Ala Asn Lys 810 815 820 gcg gcg ttc gac gcggcg agc agc ggc tgg tac aac gcc ggc ggg ggg 4171 Ala Ala Phe Asp Ala AlaSer Ser Gly Trp Tyr Asn Ala Gly Gly Gly 825 830 835 ctc gtt gtg gcc aaggcg gcg agc agt tcg gtg aac acc gcc aag acc 4219 Leu Val Val Ala Lys AlaAla Ser Ser Ser Val Asn Thr Ala Lys Thr 840 845 850 855 ttc tcg ttc acgctc ggt gag gag tcg gtc tgg gcg acg ttc tcc tgc 4267 Phe Ser Phe Thr LeuGly Glu Glu Ser Val Trp Ala Thr Phe Ser Cys 860 865 870 gag aac gcc acgacg acc ttc ggt cag tca gtg tac gtc gtc gga aat 4315 Glu Asn Ala Thr ThrThr Phe Gly Gln Ser Val Tyr Val Val Gly Asn 875 880 885 gtt ccg cag ctcggc aac tgg tcg ccg gcg gat gcc gtg aag ctc gag 4363 Val Pro Gln Leu GlyAsn Trp Ser Pro Ala Asp Ala Val Lys Leu Glu 890 895 900 ccg agc gcc tacccc acc tgg acc ggg gtg gtg cgg aac ctg ccg ccg 4411 Pro Ser Ala Tyr ProThr Trp Thr Gly Val Val Arg Asn Leu Pro Pro 905 910 915 tcg agc acg gtcgaa tgg aag tgc atc aaa cgt cag gag gcc ggc ctg 4459 Ser Ser Thr Val GluTrp Lys Cys Ile Lys Arg Gln Glu Ala Gly Leu 920 925 930 935 ccg aac acggcg gat gcg tgg gag ccc ggc ggg aac aac atc ctc tcg 4507 Pro Asn Thr AlaAsp Ala Trp Glu Pro Gly Gly Asn Asn Ile Leu Ser 940 945 950 acg cca ccttcc ggc tcg gcg ggg ata acc acc ggc gcc ttc tga 4552 Thr Pro Pro Ser GlySer Ala Gly Ile Thr Thr Gly Ala Phe 955 960 965 cccagggggg ctcgatcccggtcgccagcg caagcgcggc gcccggggtc gacgcgtgtt 4612 aggccagtac gcgaaggaaccagccctcta cgacaccggc ctcgaccccg ccgaaggact 4672 ctggcaccgg tcaggctggatcggacaaca ctgacacgcc ccgacgccat ccactctttt 4732 tggcctacaa cccgttgtcgcacgtgcgcc tcttggcccg ggcacgacga aacccccgcg 4792 atccagggat cggcgggggtttcggatggc ggtgacggtg ggatttgaac ccacggtagg 4852 gggttaccct acacaacttttcgagagttg caccttcggc cgctcggaca cgtcaccggg 4912 gtcgagttta cgcgacgttctcctggcgcg ccaatcggcg gcgccccgcc cgcgagaatc 4972 caggcccgcg ccgagaatccgcgggcgcct ggattctcag cacggggatg gattctcgcc 5032 gctcatccga gccccgcggcgagcgggctc agtgctcgtc ctccatgagc atgccgaccg 5092 aggtggcgca ggcgtcgccgcgccaggcct cgatgccctc gcgcacggcg aaggcggcga 5152 tgatgaggcc ggtgatcgcgtcggcccacc accagcccag gaggctgttg agcacgaggc 5212 ccgcgagcac ggccgccgacaggtaggtgc agatgagggt ctgcttcgag tcggccacgg 5272 cggtggccga tccgagctcgcggccggcgc ggcgctcggc gaacgacagg aacggcatga 5332 tcgccacgct gagcgccgtgatgacgatgc cgagcgtcga gtgctccacg tccgcgccgc 5392 cgacgagggc caggaccgacgtgacggtga cgtacgcggc gagcgcgaag aaggccacgg 5452 cgatgacgcg cagcgtgccgcgctcccagc gctccgggtc gcgccgcgtg aactgccacg 5512 cgacggcggc ggccgagagcacctcgatgg tcgagtccag gccgaacgcg acgagcgcgg 5572 ccgacgaggc cgcagctcccgcggcgatcg cgacgaccgc ctcgacgacg ttataggcga 5632 tggtcgcggc gacgatccagcggatgcgcc gctgcaggac ggatcgccga tcggcagacg 5692 cggtggcggt catgcgcaggtgcagctctc tccggcgcag cagccgggct cgacgtacag 5752 gacgacgcgc agcagctcgtcgagcgcggg cgcgaggtgg gcgtcggcca gccggtacc 5811

1. A polypeptide which has an enzymatic activity of forming a saccharidewith a glucose polymerization degree of 3 or higher and bearing both theα-1,6 glucosidic linkage as a linkage at the non-reducing end and theα-1,4 glucosidic linkage other than the linkage at the non-reducing endfrom a saccharide with a glucose polymerization degree of 2 or higherand bearing the α-1,4 glucosidic linkage as a linkage at thenon-reducing end by α-glucosyl transferring reaction withoutsubstantially increasing the reducing power, and which comprises anamino acid sequence of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3, or anamino acid sequence having deletion, replacement, or addition of one ormore amino acid residues of SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3. 2.The polypeptide of claim 1, which has the following physicochemicalproperties: (1) Molecular weight About 74,000 to 160,000 daltons whendetermined on SDS-PAGE; (2) Optimum temperature About 40° C. to 50° C.when incubated at pH 6.0 for 60 minutes; About 45° C. to 55° C. whenincubated at pH 6.0 for 60 minutes in the presence of 1 mM Ca²⁺; About60° C. when incubated at pH 8.4 for 60 minutes; or About 65° C. whenincubated at pH 8.4 for 60 minutes in the presence of 1 mM Ca²⁺; (3)Optimum pH About 6.0 to 8.4 when incubated at 35° C. for 60 minutes; (4)Thermal stability About 45° C. or lower when incubated at pH 6.0 for 60minutes; About 60° C. or lower when incubated at pH 8.0 for 60 minutesin the presence of 1 mM Ca²⁺; About 55° C. or lower when incubated at pH8.0 for 60 minutes; or About 60° C. or lower when incubated at pH 8.0for 60 minutes in the presence of 1 mM Ca²⁺; (5) pH Stability About 5.0to 10.0 when incubated at 4° C. for 24 hours.
 3. A DNA, which encodesthe polypeptide of claim 1 or
 2. 4. The DNA of claim 3, which comprisesa nucleotide sequence of SEQ ID NO:4, SEQ ID NO;5,or SEQ ID NO:6, or anucleotide sequence having deletion, replacement, or insertion of one ormore nucleotides of SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:6, acomplementary nucleotide sequences thereof; or a complementarynucleotide sequence in which one or more nucleotides are replaced withother nucleotide(s) based on the genetic code degeneracy withoutchanging the amino acid sequence encoded thereby.
 5. The DNA of claim 3or 4, which is obtainable by replacing one or more nucleotides of SEQ IDNO:4, SEQ ID NO:5, or SEQ ID NO:6 with other nucleotides withoutchanging the amino acid sequence of SEQ ID NO:1, SEQ ID NO:2, or SEQ IDNO:3 based on the genetic code degeneracy.
 6. The DNA of any one ofclaims 3 to 5, which is originated from a microorganism of the genusBacillus.
 7. The DNA of any one of claims 3 to 5, which is originatedfrom a microorganism of the genus Arthrobacter.
 8. A replicablerecombinant DNA, which comprises the DNA of any one of claims 3 to 7 andan autonomously replicable vector.
 9. The replicable recombinant DNA ofclaim 8, wherein said autonomously-replicable vector is a plasmidvector, Bluescript II SK(+).
 10. A transformant, which is constructed byintroducing the recombinant DNA of claim 8 or 9 into an appropriatehost.
 11. The transformant of claim 10, wherein said host is amicroorganism of the species Escherichia coli.
 12. A process forproducing the polypeptide of claim 1 or 2, which comprises the steps ofculturing the transformant of claim 10 or 11 to produce the polypeptideand collecting the polypeptide from the resulting culture.
 13. Theprocess of claim 12, wherein the polypeptide of claim 1 or 2 iscollected by one or more techniques selected from the group consistingof centrifuge, filtration, concentration, salting out, dialysis,concentration, separatory precipitation, ion-exchange chromatography,gel filtration chromatography, hydrophobic chromatography, affinitychromatography, gel electrophoresis, and isoelectric focusing.
 14. Amethod for producing a saccharide with a glucose polymerization degreeof 3 or higher and bearing both the α-1,6 glucosidic linkage as alinkage at the non-reducing end and the α-1,4 glucosidic linkage otherthan the linkage at the non-reducing end, which comprises a step ofallowing the polypeptide of claim 1 or 2 to act on a saccharide with aglucose polymerization degree of 2 or higher and bearing the α-1,4glucosidic linkage as a linkage at the non-reducing end by α-glucosyltransferring reaction without substantially increasing the reducingpower.
 15. A process of producing a saccharide with a glucosepolymerization degree of 3 or higher and bearing both the α-1,6glucosidic linkage as a linkage at the non-reducing end and the α-1,4glucosidic linkage other than the linkage at the non-reducing end, whichcomprises a step of allowing the polypeptide of claim 1 or 2 to act on asaccharide with a glucose polymerization degree of 2 or higher andbearing the α-1,4 glucosidic linkage as a linkage at the non-reducingend by α-glucosyl transferring reaction without substantially increasingthe reducing power.
 16. A process for producing a cyclotetrasaccharidehaving the structure ofcyclo{→6)-α-D-glucopyranosyl-(1→3)-α-D-glucopyranosyl-(1→6)-α-D-glucopyranosyl-(1→3)-α-D-glucopyranosyl-(1→},which comprises the steps of forming a saccharide with a glucosepolymerization degree of 3 or higher and bearing both the α-1,6glucosidic linkage as a linkage at the non-reducing end and the α-1,4glucosidic linkage other than the linkage at the non-reducing end byallowing the polypeptide of claim 1 or 2 to act on a saccharide with aglucose polymerization degree of 2 or higher and bearing the α-1,4glucosidic linkage as a linkage at the non-reducing end by α-glucosyltransferring reaction without substantially increasing the reducingpower, sequentially forming the cyclotetrasaccharide by allowing anenzyme having an activity of forming a cyclotetrasaccharide having thestructure ofcyclo{6)-α-D-glucopyranosyl-(1→3)-α-D-glucopyranosyl-(1→6)-α-D-glucopyranosyl-(1→3)-α-D-glucopyranosyl-(1→}from a saccharide with a glucose polymerization degree of 3 or higherand bearing both the α-1,6 glucosidic linkage as a linkage at thenon-reducing end and the α-1,4 glucosidic linkage other than the linkageat the non-reducing end by α-isomaltosyl-transferring reaction to act onthe resulting saccharide, and collecting the cyclotetrasaccharide. 17.The process of claim 16, which comprises a step of crystallizing saidcyclotetrasaccharide.
 18. The process of claim 16 or 17, wherein saidcyclotetrasaccharide is in a syrupy form or a crystalline form.